Tie ligand homologues

ABSTRACT

The present invention concerns isolated nucleic acid molecules encoding the novel TIE ligands NL1, NL5 and NL8, the proteins encoded by such nucleic acid molecules, as well as methods and means for making and using such nucleic acid and protein molecules.

This is a divisional of application(s) Ser. No. 08/933,821 filed on Sep.19, 1997, and issued as U.S. Pat. No. 5,972,338 which application isincorporated herein by reference and to which application(s) priority isclaimed under 35 USC §120.

FIELD OF THE INVENTION

The present invention concerns isolated nucleic acid molecules encodingnovel TIE ligand homologue, the TIE proteins encoded by such nucleicacid molecules, as well as methods and means for making and using suchnucleic acid and protein molecules.

BACKGROUND ART

The abbreviations “TIE” or “tie” are acronyms, which stand for “tyrosinekinase containing Ig and EGF homology domains” and were coined todesignate a new family of receptor tyrosine kinases which are almostexclusively expressed in vascular endothelial cells and earlyhemopoietic cells, and are characterized by the presence of an EGF-likedomain, and extracellular folding units stabilized by intra-chaindisulfide bonds, generally referred to as “immunoglobulin (IG)-like”folds. A tyrosine kinase homologous cDNA fragment from human leukemiacells (tie) was described by Partanen et al., Proc. Natl. Acad. Sci. USA87, 8913-8917 (1990). The mRNA of this human “tie” receptor has beendetected in all human fetal and mouse embryonic tissues, and has beenreported to be localized in the cardiac and vascular endothelial cells.Korhonen et al., Blood 80, 2548-2555 (1992); PCT Application PublicationNo. WO 93/14124 (published Jul. 22, 1993). The rat homolog of human tie,referred to as “tie-1”, was identified by Maisonpierre et al., Oncogene8, 1631-1637 (1993)). Another tie receptor, designated “tie-2” wasoriginally identified in rats (Dumont et al., Oncogene 8, 1293-1301(1993)), while the human homolog of tie-2, referred to as “ork” wasdescribed in U.S. Pat. No. 5,447,860 (Ziegler). The murine homolog oftie-2 was originally termed “tek.” The cloning of a mouse tie-2 receptorfrom a brain capillary cDNA library is disclosed in PCT ApplicationPublication No. WO 95/13387 (published May 18, 1995). The TIE receptorsare believed to be actively involved in angiogenesis, and may play arole in hemopoiesis as well.

The expression cloning of human TIE-2 ligands has been described in PCTApplication Publication No. WO 96/11269 (published Apr. 18, 1996) and inU.S. Pat. No. 5,521,073 (published May 28, 1996). A vector designated asλgt10 encoding a TIE-2 ligand named “htie-2 ligand 1” or “hTL1” has beendeposited under ATCC Accession No. 75928. A plasmid encoding anotherTIE-2 ligand designated “htie-2 2” or “hTL2” is available under ATCCAccession No. 75928. This second ligand has been described as anantagonist of the TIE-2 receptor. The identification of secreted humanand mouse ligands for the TIE-2 receptor has been reported by Davis etal., Cell 87, 1161-1169 (1996). The human ligand designated“Angiopoietin-1”, to reflect its role in angiogenesis and potentialaction during hemopoiesis, is the same ligand as the ligand variouslydesignated as “htie-2 1“or “hTL-1” in WO 96/11269. Angiopoietin-1 hasbeen described to play an angiogenic role later and distinct from thatof VEGF (Suri et al., Cell 87, 1171-1180 (1996)). Since TIE-2 isapparently upregulated during the pathologic angiogenesis requisite fortumor growth (Kaipainen et al., Cancer Res. 54 6571-6577 (1994))angiopoietin-1 has been suggested to be additionally useful forspecifically targeting tumor vasculature (Davis et al., supra).

SUMMARY OF THE INVENTION

The present invention concerns novel human TIE ligand homologues withpowerful effects on vasculature. The invention also provides forisolated nucleic acid molecules encoding such ligands or functionalderivatives thereof, and vectors containing such nucleic acid molecules.The invention further concerns host cells transformed with such nucleicacid to produce the novel TIE ligand homologues or functionalderivatives thereof. The novel ligands may be agonists or antagonists ofTIE receptors, known or hereinafter discovered. Their therapeutic ordiagnostic use, including the delivery of other therapeutic ordiagnostic agents to cells expressing the respective TIE receptors, isalso within the scope of the present invention.

The present invention further provides for agonist or antagonistantibodies specifically binding the TIE ligand homologues herein, andthe diagnostic or therapeutic use of such antibodies.

In another aspect, the invention concerns compositions comprising thenovel ligand homologues or antibodies.

In a further aspect, the invention concerns conjugates of the novel TIEligand homologues of the present invention with other therapeutic orcytotoxic agents, and compositions comprising such conjugates. Becausethe TIE-2 receptor has been reported to be upregulated during thepathologic angiogenesis that is requisite for tumor growth, theconjugates of the TIE ligands of the present invention to cytotoxic orother anti-tumor agents are useful in specifically targeting tumorvasculature.

In yet another aspect, the invention concerns a method for identifying acell that expresses a TIE (e.g. TIE-2) receptor, which comprisescontacting a cell with a detectably labeled TIE ligand homologues of thepresent invention under conditions permitting the binding of such TIEligand to the TIE receptor, and determining whether such binding hasindeed occurred.

In a different aspect, the invention concerns a method for measuring theamount of a TIE ligand homologues of the present invention in abiological sample by contacting the biological sample with at least oneantibody specifically binding the TIE ligand, and measuring the amountof the TIE ligand-antibody complex formed.

The invention further concerns a screening method for identifyingpolypeptide or small molecule agonists or antagonists of a TIE receptorbased upon their ability to compete with a native or variant TIE ligandof the present invention for binding to a corresponding TIE receptor.

The invention also concerns a method for imaging the presence ofangiogenesis in wound healing, in inflammation or in tumors of humanpatients, which comprises administering detectably labeled TIE ligandsor agonist antibodies of the present invention, and detectingangiogenesis.

In another aspect, the invention concerns a method of promoting,orinhibiting neovascularization in a patient by administering an effectiveamount of a TIE ligand homologues of the present invention in apharmaceutically acceptable vehicle. In a preferred embodiment, thepresent invention concerns a method for the promotion of wound healing.In another embodiment, the invention concerns a method for promotingangiogenic processes, such as for inducing collateral vascularization inan ischemic heart or limb. In a further preferred embodiment, theinvention concerns a method for inhibiting tumor growth.

In yet another aspect, the invention concerns a method of promoting bonedevelopment and/or maturation and/or growth in a patient, comprisingadministering to the patient an effective amount of a TIE ligandhomologues of the present invention in a pharmaceutically acceptablevehicle.

In a further aspect, the invention concerns a method of promoting musclegrowth and development, which comprises administering a patient in needan effective amount of a TIE ligand homologues of the present inventionin a pharmaceutically acceptable vehicle.

The TIE ligand homologues of the present invention may be administeredalone, or in combination with each other and/or with other therapeuticor diagnostic agents, including members of the VEGF family. Combinationstherapies may lead to new approaches for promoting or inhibitingneovascularization, and muscle growth and development.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1 and 1A-2 are the nucleotide sequence of FLS139 (SEQ. ID. NO.:16).

FIGS. 1B-1 and 1B-2 are the amino acid sequence of FLS139 (SEQ. ID. NO.:17).

FIGS. 2A and 2B are the nucleotide sequence of the TIE ligand NL1 (SEQ.ID. NO: 1) (DNA 22779).

FIGS. 3A and 3B are the amino acid sequence of the TIE ligand NL1 (SEQ.ID. NO:2).

FIGS. 4A and 4C are the nucleotide sequence of the TIE ligand NL5 (SEQ.ID. NO: 3) (DNA 28497).

FIGS. 5A and 5B are the amino acid sequence of the TIE ligand NL5 (SEQ.ID. NO: 4).

FIGS. 6A and 6B are the nucleotide sequence of the TIE ligand NL8 (SEQ.ID NO: 5) (DNA 23339).

FIGS. 7A and 7B are the amino acid sequence of the TIE ligand NL8 (SEQ.ID NO:6).

FIGS. 8A and 8B show the expression of NL1 in various tissues asdetermined by in situ hybridization to cellular RNA.

FIGS. 9A and 9B show the expression of NL5 in various tissues asdetermined by in situ hybridization to cellular RNA.

FIGS. 10A and 10B show the expression of NL8 in various tissues asdetermined by in situ hyridization to cellular RNA.

FIGS. 11 and 12—Northern blots showing the expression of the mRNAs ofTIE ligand homologues NL1 and NL5 in various tissues.

DETAILED DESCRIPTION OF THE INVENTION A. TIE LIGAND HOMOLOGUES ANDNUCLEIC ACID MOLECULES ENCODING THEM

The TIE ligand homologues of the present invention include the nativehuman ligands designated NL1 (SEQ. ID. NO: 2), NL5 (SEQ. ID. NO: 4), andNL8 (SEQ. ID. NO: 6), their homologs in other, non-human mammalianspecies, including, but not limited to, higher mammals, such as monkey;rodents, such as mice, rats, hamster; porcine; equine; bovine; naturallyoccurring allelic and splice variants, and biologically active(functional) derivatives, such as, amino acid sequence variants of suchnative molecules, as long as they differ from a native TL-1 or TL-2ligand. The native TIE ligand homologues of the present invention aresubstantially free of other proteins with which they are associated intheir native environment. This definition is not limited in any way bythe method(s) by which the TIE ligand homologues of the presentinvention are obtained, and includes all ligands otherwise within thedefinition, whether purified from natural source, obtained byrecombinant DNA technology, synthesized, or prepared by any combinationof these and/or other techniques. The amino acid sequence variants ofthe native TIE ligand homologues of the present invention shall have atleast about 90%, preferably, at least about 95%, more preferably atleast about 98%, most preferably at least about 99% sequence identitywith a full-length, native human TIE ligand homologues of the presentinvention, or with the fibrinogen-like domain of a native human TIEligand homologues of the present invention. Such amino acid sequencevariants preferably exhibit or inhibit a qualitative biological activityof a native TIE ligand homologues.

The term “fibrinogen domain” or “fibrinogen-like domain” is used torefer to amino acids from about position 278 to about position 498 inthe known hTL-1 amino acid sequence; amino acids from about position 276to about position 496 in the known hTL,2 amino acid sequence; aminoacids from about position 270 to about 493 in the amino acid sequence ofNL1; amino acids from about position 272 to about position 491 in theamino acid sequence of NL5; and amino acids from about position 252 toabout position 470 in the amino acid sequence of NL8; and to homologousdomains in other TIE ligands.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given TIE ligandhomologues may be produced. The present invention specificallycontemplates every possible variation of nucleotide sequences, encodingthe TIE ligand homologues of the present invention, based upon allpossible codon choices. Although nucleic acid molecules which encode theTIE ligand homologues herein are preferably capable of hybridizing,under stringent conditions, to a naturally occurring TIE ligandhomologues gene, it may be advantageous to produce nucleotide sequencesencoding TIE ligand homologues, which possess a substantially differentcodon usage. For example, codons may be selected to increase the rate atwhich expression of the polypeptide occurs in a particular prokaryoticor eukaryotic host cells, in accordance with the frequency with which aparticular codon is utilized by the host. In addition, RNA transcriptswith improved properties, e.g. half-life can be produced by properchoice of the nucleotide sequences encoding a given TIE ligandhomologues.

“Sequence identity” shall be determined by aligning the two sequences tobe compared following the Clustal method of multiple sequence alignment(Higgins et al., Comput. Appl. Biosci. 5, 151-153 (1989), and Higgins etal., Gene 73, 237-244 (1988)) that is incorporated in version 1.6 of theLasergene biocomputing software (DNASTAR, Inc., Madison, Wis.), or anyupdated version or equivalent of this software.

The terms “biological activity” and “biologically active” with regard toa TIE ligand of the present invention refer to the ability of a moleculeto specifically bind to and signal through a native TIE receptor, e.g. anative TIE-2 receptor, or to block the ability of a native TIE receptor(e.g. TIE-2) to participate in signal transduction. Thus, the (nativeand variant) TIE ligand homologues of the present invention includeagonists and antagonists of a native TIE, e.g. TIE-2, receptor.Preferred biological activities of the TIE ligand homologues of thepresent invention include the ability to induce or inhibitvascularization. The ability to induce vascularization will be usefulfor the treatment of biological conditions and diseases, wherevascularization is desirable, such as wound healing, ischaemia, anddiabetes. On the other hand, the ability to inhibit or blockvascularization may, for example, be useful in preventing or attenuatingtumor growth. Another preferred biological activity is the ability toaffect muscle growth or development. A further preferred biologicalactivity is the ability to influence bone development, maturation, orgrowth.

The term “functional derivative” is used to define biologically activeamino acid sequence variants of the native TIE ligand homologues of thepresent invention, as well as covalent modifications, includingderivatives obtained by reaction with organic derivatizing agents,post-translational modifications, derivatives with nonproteinaceouspolymers, and immunoadhesins.

“Vascular endothelial growth factor”/“vascular permeability factor”(VEGF/VPF) is an endothelial cell-specific mitogen which has recentlybeen shown to be stimulated by hypoxia and required for tumorangiogenesis (Senger et al., Cancer 46: 5629-5632 (1986); Kim et al.,Nature 362:841-844 (1993); Schweiki et al., Nature 359: 843-845 (1992);Plate et al., Nature 359: 845-848 (1992)). It is a 34-43 kDa (with thepredominant species at about 45 kDa) dimeric, disulfide-linkedglycoprotein synthesized and secreted by a variety of tumor and normalcells. In addition, cultured human retinal cells such as pigmentepithelial cells and pericytes have been demonstrated to secrete VEGFand to increase VEGF gene expression in response to hypoxia (Adamis etal., Biochem. Biophys. Res. Commun. 193: 631-638 (1993); Plouet et al.,Invest. Ophthalmol. Vis. Sci. 34: 900 (1992); Adamis et al., Invest.Ophthalmol. Vis. Sci. 34: 1440 (1993); Aiello et al., Invest. Opthalmol.Vis. Sci. 35: 1868 (1994); Simorre-pinatel et al., Invest. Opthalmol.Vis. Sci. 35: 3393-3400 (1994)). In contrast, VEGF in normal tissues isrelatively low. Thus, VEGF appears to play a principle role in manypathological states and processes related to neovascularization.Regulation of VEGF expression in tissues affected by the variousconditions described above could therefore be key in treatment orpreventative therapies associated with hypoxia.

The term “isolated” when used to describe the various polypeptidesdescribed herein, means polypeptides that have been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the TIE ligand'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes nucleic acid molecules contained in cells that ordinarilyexpress an TIE ligand of the present invention, where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.Deletional variants include those having C- and/or N-terminal deletions(truncations) as well as variants with internal deletions of one or moreamino acids. The preferred deletional variants of the present inventioncontain deletions outside the fibrinogen-like domain of a native TIEligand homologues of the present invention.

The amino acid sequence variants of the present invention may containvarious combinations of amino acid substitutions, insertions and/ordeletions, to produce molecules with optimal characteristics.

The amino acids may be classified according to the chemical compositionand properties of their side chains. They are broadly classified intotwo groups, charged and uncharged. Each of these groups is divided intosubgroups to classify the amino acids more accurately.

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions areexpected to result in significant changes in the biologicalproperties/function of the obtained variant

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions not directlyinvolved in the interaction with a native TIE receptor. Deletions arepreferably performed outside the fibrinogen-like regions at theC-terminus of the TIE ligand homologues of the present invention.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the TIE ligand homologues amino acid sequence) may rangegenerally from about 1 to 10 residues, more preferably 1 to 5 residues,more preferably 1 to 3 residues. Examples of terminal insertions includethe TIE ligand homologues with an N-terminal methionyl residue, anartifact of its direct expression in bacterial recombinant cell culture,and fusion of a heterologous N-terminal signal sequence to theN-terminus of the TIE ligand homologues molecule to facilitate thesecretion of the mature TIE ligand homologues from recombinant hostcells. Such signal sequences will generally be obtained from, and thushomologous to, the intended host cell species. Suitable sequencesinclude, for example, STII or Ipp for E. coli, alpha factor for yeast,and viral signals such as herpes gD for mammalian cells. Otherinsertional variants of the native TIE ligand homologues moleculesinclude the fusion of the N- or C-terminus of the TIE ligand homologuesmolecule to immunogenic polypeptides, e.g. bacterial polypeptides suchas beta-lactamase or an enzyme encoded by the E. coli trp locus, oryeast protein, and C-terminal fusions with proteins having a longhalf-life such as immunoglobulin regions (preferably immunoglobulinconstant regions), albumin, or ferritin, as described in WO 89/02922published on Apr. 6, 1989.

Since it is often difficult to predict in advance the characteristics ofa variant TIE ligand homologues, it will be appreciated that somescreening will be needed to select the optimum variant.

Amino acid sequence variants of native TIE ligand homologues of thepresent invention are prepared by methods known in the art byintroducing appropriate nucleotide changes into a native or variant TIEligand homologues DNA, or by in vitro synthesis of the desiredpolypeptide. There are two principal variables in the construction ofamino acid sequence variants: the location of the mutation site and thenature of the mutation. With the exception of naturally-occurringalleles, which do not require the manipulation of the DNA sequenceencoding the TIE ligand homologues, the amino acid sequence variants ofTIE are preferably constructed by mutating the DNA, either to arrive atan allele or an amino acid sequence variant that does not occur innature.

One group of the mutations will be created within the domain or domainsof the TIE ligand homologues of the present invention identified asbeing involved in the interaction with a TIE receptor, e.g. TIE-1 orTIE-2.

Alternatively or in addition, amino acid alterations can be made atsites that differ in TIE ligand homologues from various species, or inhighly conserved regions, depending on the goal to be achieved.

Sites at such locations will typically be modified in series, e.g. by(1) substituting first with conservative choices and then with moreradical selections depending upon the results achieved, (2) deleting thetarget residue or residues, or (3) inserting residues of the same ordifferent class adjacent to the located site, or combinations of options1-3.

One helpful technique is called “alanine scanning” (Cunningham andWells, Science 244, 1081-1085 [1989]). Here, a residue or group oftarget residues is identified and substituted by alanine or polyalanine.Those domains demonstrating functional sensitivity to the alaninesubstitutions are then refined by introducing further or othersubstituents at or for the sites of alanine substitution.

After identifying the desired mutation(s), the gene encoding an aminoacid sequence variant of a TIE ligand homologues can, for example, beobtained by chemical synthesis as hereinabove described.

More preferably, DNA encoding a TIE ligand homologues amino acidsequence variant is prepared by site-directed mutagenesis of DNA thatencodes an earlier prepared variant or a nonvariant version of theligand. Site-directed (site-specific) mutagenesis allows the productionof ligand variants through the use of specific oligonucleotide sequencesthat encode the DNA sequence of the desired mutation, as well as asufficient number of adjacent nucleotides, to provide a primer sequenceof sufficient size and sequence complexity to form a stable duplex onboth sides of the deletion junction being traversed. Typically, a primerof about 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the techniques of site-specific mutagenesis are well known inthe art, as exemplified by publications such as, Edelman et al., DNA 2,183 (1983). As will be appreciated, the site-specific mutagenesistechnique typically employs a phage vector that exists in both asingle-stranded and double-stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage, forexample, as disclosed by Messing et al., Third Cleveland Symposium onMacromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam(1981). This and other phage vectors are commercially available andtheir use is well known to those skilled in the art. A versatile andefficient procedure for the construction of oligodeoxyribonucleotidedirected site-specific mutations in DNA fragments using M13-derivedvectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res.10, 6487-6500 [1982]). Also, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153, 3 [1987]) may be employed to obtain single-stranded DNA.Alternatively, nucleotide substitutions are introduced by synthesizingthe appropriate DNA fragment in vitro, and amplifying it by PCRprocedures known in the art.

In general, site-specific mutagenesis herewith is performed by firstobtaining a single-stranded vector that includes within its sequence aDNA sequence that encodes the relevant protein. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., Proc. Natl.Acad. Sci. USA 75, 5765 (1978). This primer is then annealed with thesingle-stranded protein sequence-containing vector, and subjected toDNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment,to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells such as JP101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region may be removed and placed in anappropriate expression vector for protein production.

The PCR technique may also be used in creating amino acid sequencevariants of a TIE ligand homologues. When small amounts of template DNAare used as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template. For introduction of a mutation into aplasmid DNA, one of the primers is designed to overlap the position ofthe mutation and to contain the mutation; the sequence of the otherprimer must be, identical to a stretch of sequence of the oppositestrand of the plasmid, but this sequence can be located anywhere alongthe plasmid DNA. It is preferred, however, that the sequence of thesecond primer is located within 200 nucleotides from that of the first,such that in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the position of the mutation specified by the primer, andpossibly at other positions, as template copying is somewhaterror-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more) partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp^(R) kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayered with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/l), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows:

2 min. 55° C.,

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.,

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. [Gene 34, 315 (1985)]. Thestarting material is the plasmid (or vector) comprising the TIE ligandDNA to be mutated. The codon(s) within the TIE ligand to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the DNA encoding the TIE ligand. After therestriction sites have been introduced into the plasmid, the plasmid iscut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction site butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated TIE ligand DNA sequence.

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant TIE ligandhomologues. This method involves (a) constructing a replicableexpression vector comprising a first gene encoding an receptor to bemutated, a second gene encoding at least a portion of a natural orwild-type phage coat protein wherein the first and second genes areheterologous, and a transcription regulatory element operably linked tothe first and second genes, thereby forming a gene fusion encoding afusion protein; (b) mutating the vector at one or more selectedpositions within the first gene thereby forming a family of relatedplasmids; (c) transforming suitable host cells with the plasmids; (d)infecting the transformed host cells with a helper phage having a geneencoding the phage coat protein; (e) culturing the transformed infectedhost cells under conditions suitable for forming recombinant phagemidparticles containing at least a portion of the plasmid and capable oftransforming the host, the conditions adjusted so that no more than aminor amount of phagemid particles display more than one copy of thefusion protein on the surface of the particle; (f) contacting thephagemid particles with a suitable antigen so that at least a portion ofthe phagemid particles bind to the antigen; and (g) separating thephagemid particles that bind from those that do not. Steps (d) through(g) can be repeated one or more times. Preferably in this method theplasmid is under tight control of the transcription regulatory element,and the culturing conditions are adjusted so that the amount or numberof phagemid particles displaying more than one copy of the fusionprotein on the surface of the particle is less than about 1%. Also,preferably, the amount of phagemid particles displaying more than onecopy of the fusion protein is less than 10% of the amount of phagemidparticles displaying a single copy of the fusion protein. Mostpreferably, the amount is less than 20%. Typically in this method, theexpression vector will further contain a secretory signal sequence fusedto the DNA encoding each subunit of the polypeptide and thetranscription regulatory element will be a promoter system. Preferredpromoter systems are selected from lac Z, λ_(PL), tac, T7 polymerase,tryptophan, and alkaline phosphatase promoters and combinations thereof.Also, normally the method will employ a helper phage selected fromM13K07, M13R408, M13-VCS, and Phi X 174. The preferred helper phage isM13K07, and the preferred coat protein is the M13 Phage gene III coatprotein. The preferred host is E. coli, and protease-deficient strainsof E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,Molecular Cloning: A laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and Current Protocols in Molecular Biology,Ausubel et al., eds., Wiley-Interscience, 1991.

“Immunoadhesins” are chimeras which are traditionally constructed from areceptor sequence linked to an appropriate immunoglobulin constantdomain sequence (immunoadhesins). Such structures are well known in theart. Immunoadhesins reported in the literature include fusions of the Tcell receptor* [Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,2936-2940 (1987)]; CD4* [Capon et al., Nature 337, 525-531 (1989);Traunecker et al., Nature 339, 68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9, 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990)];L-selectin (homing receptor) [Watson et al., J. Cell. Biol. 110,2221-2229 (1990); Watson et al., Nature 349, 164-167 (1991)]; CD44*[Aruffo et al., Cell 61, 1303-1313 (1990)]; CD28* and B7* [Linsley etal., J. Exp. Med. 173, 721-730 (1991)]; CTLA-4* [Lisley et al., J. Exp.Med. 174, 561-569 (1991)]; CD22* [Stamenkovic et al., Cell 66. 1133-1144(1991)]; TNF receptor [Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88,10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27, 2883-2886(1991); Peppel et al., J. Exp. Med. 174, 1483-1489 (1991)]; NP receptors[Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptorα-chain* [Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)];HGF receptor [Mark, M. R. et al., 1992, J. Biol. Chem. submitted], wherethe asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

Ligand-immunoglobulin chimeras are also known, and are disclosed, forexample, in U.S. Pat. No. 5,304,640 (for L-selectin ligands); U.S. Pat.Nos. 5,316,921 and 5,328,837 (for HGF variants). These chimeras can bemade in a similar way to the construction of receptor-immunoglobulinchimeras.

Covalent modifications of the TIE ligand homologues of the presentinvention are included within the scope herein. Such modifications aretraditionally introduced by reacting targeted amino acid residues of theTIE ligand with an organic derivatizing agent that is capable ofreacting with selected sides or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-TIE ligandantibodies for immunoaffinity purification of the recombinant. Forexample, complete inactivation of the biological activity of the proteinafter reaction with ninhydrin would suggest that at least one arginyl orlysyl residue is critical for its activity, whereafter the individualresidues which were modified under the conditions selected areidentified by isolation of a peptide fragment containing the modifiedamino acid residue. Such modifications are within the ordinary skill inthe art and are performed without undue experimentation.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86[1983]), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the TIE ligand with polypeptides as well asfor cross-linking the TIE ligand polypeptide to a water insolublesupport matrix or surface for use in assays or affinity purification. Inaddition, a study of interchain cross-links will provide directinformation on conformational structure. Commonly used cross-linkingagents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides.

Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the a -amino groups of lysine,arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983)].

Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

The TIE ligands may be linked to various nonproteinaceous polymers, suchas polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes,in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. These variants, just asthe immunoadhesins of the present invention are expected to have longerhalf-lives than the corresponding native TIE ligand homologues.

The TIE ligand homologues may be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, in colloidal drug delivery systems (e.g. liposomes,albumin microspheres, microemulsions, nano-particles and nanocapsules),or in macroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences, 16th Edition, Osol, A., Ed. (1980).

The term “native TIE receptor” is used herein to refer to a TIE receptorof any animal species, including, but not limited to, humans, otherhigher primates, e.g. monkeys, and rodents, e.g. rats and mice. Thedefinition specifically includes the TIE-2 receptor, disclosed, forexample, in PCT Application Serial No. WO 95/13387 (published May 18,1995), and the endothelial cell receptor tyrosine kinase termed “TIE” inPCT Application Publication No. WO 93/14124 (published Jul. 22, 1993),and preferably is TIE-2.

B. ANTI-TIE LIGAND HOMOLOGUES ANTIBODIES

The present invention covers agonist and antagonist antibodies,specifically binding the TIE ligand homologues. The antibodies may bemonoclonal or polyclonal, and include, without limitation, matureantibodies, antibody fragments (e.g. Fab, F(ab′)₂, F_(v), etc.),single-chain antibodies and various chain combinations.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist, antagonist, andneutralizing antibodies) specifically binding a TIE ligand of thepresent invention and antibody compositions with polyepitopicspecificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-TIE ligand antibody with a constant domain (e.g.“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv), so long as they exhibit the desired biologicalactivity. See, e.g. U.S. Pat. No. 4,816,567 and Mage et al., inMonoclonal Antibody Production Techniques and Applications, pp.79-97(Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

Polyclonal antibodies to a TIE ligand homologues of the presentinvention generally are raised in animals by multiple subcutaneous (sc)or intraperitoneal (ip) injections of the TIE ligand homologues and anadjuvant. It may be useful to conjugate the TIE ligand homologues or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glytaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-TIE ligand antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal boosted with the conjugate ofthe same TIE ligand, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-TIE ligand monoclonal antibodies of the inventionmay be made using the hybridoma method first described by Kohler &Milstein, Nature 256:495 (1975), or may be made by recombinant DNAmethods [Cabilly, et al., U.S. Pat. No. 4,816,567].

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell [Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies[Kozbor, J. Immunol. 133:3001 (1984); Brodeur, et al., MonoclonalAntibody Production Techniques and Applications, pp.51-63 (MarcelDekker, Inc., New York, 1987)].

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the TIE ligandhomologues. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard, Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of an anti-TIE ligand monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a TIEligand homologues of the present invention and another antigen-combiningsite having specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a TIE ligand homologues or an immunologically reactiveportion thereof) to compete with the test sample analyte (TIE ligandhomologues) for binding with a limited amount of antibody. The amount ofTIE ligand homologues in the test sample is inversely proportional tothe amount of standard that becomes bound to the antibodies. Tofacilitate determining the amount of standard that becomes bound, theantibodies generally are insolubilized before or after the competition,so that the standard and analyte that are bound to the antibodies mayconveniently be separated from the standard and analyte which remainunbound.

Sandwich assays involve the use of two antibodies; each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255(1993); Jakobovits et al., Nature 362, 255-258 (1993).

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aparticular TIE ligand homologues, the other one is for any otherantigen, and preferably for another ligand. For example, bispecificantibodies specifically binding two different TIE ligand homologues arewithin the scope of the present invention.

Methods for making bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, and second and thirdconstant regions of an immunoglobulin heavy chain (CH2 and CH3). It ispreferred to have the first heavy chain constant region (CH1) containingthe site necessary for light chain binding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are cotransfected into a suitable host organism.This provides for great flexibility in adjusting the mutual proportionsof the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in application Ser.No. 07/931,811 filed Aug. 17, 1992.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

The term “agonist” is used to refer to peptide and non-peptide analogsof the native TIE ligand homologues of the present invention and toantibodies specifically binding such native TIE ligand homologues,provided that they have the ability to signal through a native TIEreceptor (e.g. TIE-2). In other words, the term “agonist” is defined inthe context of the biological role of the TIE receptor, and not inrelation to the biological role of a native TIE ligand homologues,which, as noted before, may be an agonist or antagonist of the TIEreceptor biological function. Preferred agonists are promoters ofvascularization.

The term “antagonist” is used to refer to peptide and non-peptideanalogs of the native TIE ligand homologues of the present invention andto antibodies specifically binding such native TIE ligand homologues,provided that they have the ability to inhibit the biological functionof a native TIE receptor (e.g. TIE-2). Again, the term “antagonist” isdefined in the context of the biological role of the TIE receptor, andnot in relation to the biological activity of a native TIE ligandhomologues, which may be either an agonist or an antagonist of the TIEreceptor biological function. Preferred antagonists are inhibitors ofvasculogenesis.

C. CLONING AND EXPRESSION OF THE TIE LIGAND HOMOLOGUES

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of the TIE ligand molecule that is inserted into thevector. If the signal sequence is heterologous, it should be selectedsuch that it is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell.

Heterologous signal sequences suitable for prokaryotic host cells arepreferably prokaryotic signal sequences, such as the α-amylase, ompA,ompC, ompE, ompF, alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the yeastinvertase, amylase, alpha factor, or acid phosphatase leaders may, forexample, be used. In mammalian cell expression mammalian signalsequences are most suitable. The listed signal sequences are forillustration only, and do not limit the scope of the present inventionin any way.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenabled the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequence are well known for a variety of bacteria, yeast and viruses.The origin of replication from the well-known plasmid pBR322 is suitablefor most gram negative bacteria, the 2 μ plasmid origin for yeast andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins of replication are notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter). Most expressionvectors are “shuttle” vectors, i.e. they are capable of replication inat least one class of organisms but can be transfected into anotherorganism for expression. For example, a vector is cloned in E. coli andthen the same vector is transfected into yeast or mammalian cells forexpression even though it is not capable of replicating independently ofthe host cell chromosome.

DNA is also cloned by insertion into the host genome. This is readilyaccomplished using Bacillus species as hosts, for example, by includingin the vector a DNA sequence that is complementary to a sequence foundin Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theDNA encoding the desired heterologous polypeptide. However, the recoveryof genomic DNA is more complex than that of an exogenously replicatedvector because restriction enzyme digestion is required to excise theencoded polypeptide molecule.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forbacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin [Southern et al., J. Molec. Appl. Genet. 1, 327(1982)], mycophenolic acid [Mulligan et al., Science 209, 1422 (1980)],or hygromycin [Sudgen et al., Mol. Cel. Biol. 5, 410-413 (1985)]. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Other examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the desirednucleic acid. The mammalian cell transform ants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the desired polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the desiredpolypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumwhich lacks hypoxanthine, glycine, and thymidine. An appropriate hostcell in this case is the Chinese hamster ovary (CHO) cell line deficientin DHFR activity, prepared and propagated as described by Urlaub andChasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). A particularlyuseful DHFR is a mutant DHFR that is highly resistant to MTX (EP117,060). This selection agent can be used with any otherwise suitablehost, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR. The DNA encoding DHFR and the desired polypeptide,respectively, then is amplified by exposure to an agent (methotrexate,or MTX) that inactivates the DHFR. One ensures that the cell requiresmore DHFR (and consequently amplifies all exogenous DNA) by selectingonly for cells that can grow in successive rounds of ever-greater MTXconcentration. Alternatively, hosts co-transformed with genes encodingthe desired polypeptide, wild-type DHFR, and another selectable markersuch as the neo gene can be identified using a selection agent for theselectable marker such as G418 and then selected and amplified usingmethotrexate in a wild-type host that contains endogenous DHFR. (Seealso U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282:39; Kingsmanet al., 1979, Gene 7:141; or Tschemper et al., 1980, Gene 10: 157). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors, unlike cloning vectors, should contain a promoterwhich is recognized by the host organism and is operably linked to thenucleic acid encoding the desired polypeptide.

Promoters are untranslated sequences located upstream from the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of nucleic acid under theircontrol. They typically fall into two classes, inducible andconstitutive. Inducible promoters are promoters that initiate increasedlevels of transcription from DNA under their control in response to somechange in culture conditions, e.g. the presence or absence of a nutrientor a change in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known. Thesepromoters are operably linked to DNA encoding the desired polypeptide byremoving them from their gene of origin by restriction enzyme digestion,followed by insertion 5′ to the start codon for the polypeptide to beexpressed. This is not to say that the genomic promoter for a TIE ligandis not usable. However, heterologous promoters generally will result ingreater transcription and higher yields of expressed TIE ligands ascompared to the native TIE ligand promoters.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes. 8:4057 (1980) and EPO Appln. Publ. No. 36,776) and hybrid promoterssuch as the tac promoter (H. de Boer et al., Proc. Nat'l. Acad. Sci. USA80:21-25 (1983)). However, other known bacterial promoters are suitable.Their nucleotide sequences have been published, thereby enabling askilled worker operably to ligate them to DNA encoding a TIE ligand(Siebenlist et al., Cell 20:269 (1980)) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked-to the DNA encoding a TIE ligand.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1978); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

TIE ligand homologues transcription from vectors in mammalian host cellsmay be controlled by promoters obtained from the genomes of viruses suchas polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat shock promoters, and from the promoter normally associated with theTIE ligand homologues sequence, provided such promoters are compatiblewith the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment which also contains the SV40 viralorigin of replication [Fiers et al., Nature 273:113 (1978), Mulligan andBerg, Science 209, 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA 78, 7398-7402 (1981)]. The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment [Greenaway et al., Gene 18,355-360 (1982)]. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso, Gray et al., Nature 295, 503-508 (1982) on expressing cDNAencoding human immune interferon in monkey cells; Reyes et al., Nature297, 598-601 (1982) on expressing human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79, 5166-5170 (1982)on expression of the human interferon β1 gene in cultured mouse andrabbit cells; and Gorman et al., Proc. Natl. Acad. Sci., USA 79,6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse HIN-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the TIE ligand homologues of the presentinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation and positionindependent having been found 5′ [Laimins et al., Proc. Natl. Acad. Sci.USA 78, 993 (1981)] and 3′ [Lasky et al., Mol Cel. Biol. 3, 1108 (1983)]to the transcription unit, within an intron [Banerji et al., Cell 33,729 (1983)] as well as within the coding sequence itself [Osborne etal., Mol. Cel. Biol. 4, 1293 (1984)]. Many enhancer sequences are nowknown from mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297, 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theTIE ligand homologues DNA, but is preferably located at a site 5′ fromthe promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the TIE ligand homologues. The 3′untranslated regions also include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components, the desired coding and control sequences, employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TIE ligand homologues. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded byclones DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of a TIE ligand homologues.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the TIE polypeptides in recombinant vertebrate cell cultureare described in Getting et al., Nature 293, 620-625 (1981); Mantel etal., Nature 281, 4046 (1979); Levinson et al.; EP 117,060 and EP117,058. A particularly useful plasmid for mammalian cell cultureexpression of the TIE ligand homologues polypeptides is pRK5 (EP307,247), along with its derivatives, such as, pRK5D that has an sp6transcription initiation site followed by an SfiI restriction enzymesite preceding the Xho/NotlI cDNA cloning sites, and pRK5B, a precursorof pRK5D that does not contain the SfiI site; see, Holmes et al.,Science 253, 1278-1280 (1991).

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequences by the methods of Messing et al., NucleiAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TIE ligand homologues. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high level ofa desired polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17-16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive screening of such polypeptides for desired biological orphysiological properties. Thus transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of native TIE ligand homologues with the requisitebiological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of a TIE ligand homologues (including functional derivativesof native proteins) in recombinant vertebrate cell culture are describedin Gething et al., Nature 293 620-625 (1981); Mantei et al., Nature 281,4046 (1979); Levinson et al., EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of a TIE ligand ispRK5 (EP 307,247) or pSVI6B (PCT Publication No. WO 91/08291).

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast or higher eukaryote cells described above. Suitableprokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B. E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe [Beach and Nurse, Nature 290, 140 (1981)], Kluyveromyces lactis[Louvencourt et al., J. Bacteriol. 737 (1983)]; yarrowia (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa [Case et al., Proc. Natl. Acad. Sci. USA 76, 5259-5263(1979)]; and Aspergillus hosts such as A. nidulans [Ballance et al.,Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al., Gene26, 205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA 81,1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4, 475-479(1985)].

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa california NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Generally, plant cells are transfected by incubation with certainstrains of the bacterium Agrobacterium tumefaciens, which has beenpreviously manipulated to contain the TIE ligand homologues DNA. Duringincubation of the plant cell culture with A. tumefaciens, the DNAencoding a TIE ligand homologues is transferred to the plant cell hostsuch that it is transfected, and will, under appropriate conditions,express the TIE ligand homologues DNA. In addition, regulatory andsignal sequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appyl. Gen. 1, 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known.See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cellline [293 or 293 cells subcloned for growth in suspension culture,Graham et al., J. Gen. Virol. 36, 59 (1977)]; baby hamster kidney cells9BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR [CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA 77, 4216 (1980)]; mouse sertollicells [TM4, Mather, Biol. Reprod. 23, 243-251 (1980)]; monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells [Mather et al., Annals N.Y. Acad. Sci. 383, 44068 (1982)]; MRC5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Preferredhost cells are human embryonic kidney 293 and Chinese hamster ovarycells.

Particularly preferred host cells for the purpose of the presentinvention are vertebrate cells producing the TIE ligand homologues ofthe present invention.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as is appropriate for inducingpromoters or selecting transformants containing amplified genes.

Prokaryotes cells used to produced the TIE ligand homologues of thisinvention are cultured in suitable media as describe generally inSambrook et al., supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium(DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44(1979); Barnes and Sato, Anal. Biochem. 102, 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or U.S. Pat. No. Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the TIE ligand homologues of thisinvention may be produced by homologous cells already containing DNAencoding the particular TIE ligand homologues.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al., Am. J. Clin. Pharm.75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native TIE ligand homologues polypeptide of the presentinvention, or against a synthetic peptide based on the DNA sequenceprovided herein as described further hereinbelow.

The TIE ligand homologues may be produced in host cells in the form ofinclusion bodies or secreted into the periplasmic space or the culturemedium, and is typically recovered from host cell lysates. Therecombinant ligands may be purified by any technique allowing for thesubsequent formation of a stable protein.

When the TIE ligand homologues is expressed in a recombinant cell otherthan one of human origin, it is completely free of proteins orpolypeptides of human origin. However, it is necessary to purify the TIEligand homologues from recombinant cell proteins or polypeptides toobtain preparations that are substantially homogenous as to the ligand.As a first step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The TIE ligand homologues may then be purified from thesoluble protein fraction. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; and protein A Sepharosecolumns to remove contaminants such as IgG.

Functional derivatives of the TIE ligand homologues in which residueshave been deleted, inserted and/or substituted are recovered in the samefashion as the native ligands, taking into account of any substantialchanges in properties occasioned by the alteration. For example, fusionof the TIE ligand homologues with another protein or polypeptide, e.g. abacterial or viral antigen, facilitates purification; an immunoaffinitycolumn containing antibody to the antigen can be used to absorb thefusion. Immunoaffinity columns such as a rabbit polyclonal anti-TIEligand homologues column can be employed to absorb TIE ligand homologuesvariants by binding to at least one remaining immune epitope. A proteaseinhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) also may beuseful to inhibit proteolytic degradation during purification, andantibiotics may be included to prevent the growth of adventitiouscontaminants. The TIE ligand homologues of the present invention areconveniently purified by affinity chromatography, based upon theirability to bind to a TIE receptor, e.g. TIE-2.

One skilled in the art will appreciate that purification methodssuitable for native TIE ligand homologues may require modification toaccount for changes in the character of a native TIE ligand homologuesor its variants upon expression in recombinant cell culture

D. USE OF THE TIE LIGAND HOMOLOGUES, NUCLEIC ACID MOLECULES ANDANTIBODIES

The TIE ligand homologues of the present invention are useful inpromoting the survival and/or growth and/or differentiation of TIEreceptor (e.g. TIE-2 receptor) expressing cells in cell culture.

The TIE ligand homologues may be additionally used to identify cellswhich express native TIE receptors, e.g. the TIE-2 receptor. To thisend, a detectably labeled ligand is contacted with a target cell undercondition permitting its binding to the TIE receptor, and the binding ismonitored.

The TIE ligands herein may also be used to identify molecules exhibitinga biological activity of a TIE ligand homologues, for example, byexposing a cell expressing a TIE ligand homologues herein to a testmolecule, and detecting the specific binding of the test molecule to aTIE (e.g. TIE-2) receptor, either by direct detection, or base uponsecondary biological effects. This approach is particularly suitable foridentifying new members of the TIE ligand family, or for screeningpeptide or non-peptide small molecule libraries.

The TIE ligand homologues disclosed herein are also useful in screeningassays designed to identify agonists or antagonists of a native TIE(e.g. TIE-2) receptor that play an important role in bone development,maturation or growth, or in muscle growth or development and/or promoteor inhibit angiogenesis. For example, antagonists of the TIE-2 receptormay be identified based upon their ability to block the binding of a TIEligand homologues of the present invention to a native TIE receptor, asmeasured, for example, by using BIAcore biosensor technology (BIAcore;Pharmacia Biosensor, Midscataway, N.J.); or by monitoring their abilityto block the biological response caused by a biologically active TIEligand homologues herein. Biological responses that may be monitoredinclude, for example, the phosphorylation of the TIE-2 receptor ordownstream components of the TIE-2 signal transduction pathway, orsurvival, growth or differentiation of cells expressing the TIE-2receptor. Cell-based assays, utilizing cells that do not normally theTIE-2 receptor, engineered to express this receptor, or to coexpress theTIE-2 receptor and a TIE ligand homologues of the present invention, areparticularly convenient to use.

In a particular embodiment, small molecule agonists and antagonists of anative TIE receptor, e.g. the TIE-2 receptor, can be identified, basedupon their ability to interfere with the TIE ligand/TIE receptorinteraction. There are numerous ways for measuring the specific bindingof a test molecule to a TIE receptor, including, but not limited todetecting or measuring the amount of a test molecule bound to thesurface of intact cells expressing the TIE receptor, cross-linked to theTIE receptor in cell lysates, or bound to the TIE receptor in vitro.

Detectably labeled TIE ligand homologues include, for example, TIEligand homologues covalently or non-covalently linked to a radioactivesubstances, e.g. ¹²⁵I, a fluorescent substance, a substance havingenzymatic activity (preferably suitable for colorimetric detection), asubstrate for an enzyme (preferably suitable for calorimetricdetection), or a substance that can be recognized by a(n) (detectablylabeled) antibody molecule.

The assays of the present invention may be performed in a manner similarto that described in PCT Publication WO 96/11269, published Apr. 18,1996.

The TIE ligand homologues of the present invention are also useful forpurifying TIE receptors, e.g. TIE-2 receptors, optionally used in theform of immunoadhesins, in which the TIE ligand homologues or the TIEreceptor binding portion thereof is fused to an immunoglobulin heavy orlight chain constant region.

The nucleic acid molecules of the present invention are useful fordetecting the expression of TIE ligand homologues in cells or tissuesections. Cells or tissue sections may be contacted with a detectablylabeled nucleic acid molecule encoding a TIE ligand of the presentinvention under hybridizing conditions, and the presence of mRNAhybridized to the nucleic acid molecule determined, thereby detectingthe expression of the TIE ligand homologues.

Antibodies of the present invention may, for example, be used inimmunoassays to measure the amount of a TIE ligand homologues in abiological sample. The biological sample is contacted with an antibodyor antibody mixture specifically binding the a TIE ligand homologues ofthe present invention, and the amount of the complex formed with aligand present in the test sample is measured.

Antibodies to the TIE ligand homologues herein may additionally be usedfor the delivery of cytotoxic molecules, e.g. radioisotopes or toxins,or therapeutic agents to cells expressing a corresponding TIE receptor.The therapeutic agents may, for example, be other TIE ligand homologues,including the TIE-2 ligand, members of the vascular endothelial growthfactor (VEGF) family, or known anti-tumor agents, and agents known to beassociated with muscle growth or development, or bone development,maturation, or growth.

Anti-TIE ligand homologues antibodies are also suitable as diagnosticagents, to detect disease states associated with the expression of a TIE(e.g. TIE-2) receptor. Thus, detectably labeled TIE ligand homologuesand antibody agonists of a TIE receptor can be used for imaging thepresence of angiogenesis.

In addition, the new TIE ligand homologues herein can be used to promoteneovascularization, and may be useful for inhibiting tumor growth.

Further potential therapeutic uses include the modulation of muscle andbone development, maturation, or growth.

For therapeutic use, the TIE ligand homologues or anti-TIE ligandantibodies of the present invention are formulated as therapeuticcomposition comprising the active ingredient(s) in admixture with apharmacologically acceptable vehicle, suitable for systemic or topicalapplication. The pharmaceutical compositions of the present inventionare prepared for storage by mixing the active ingredient having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as Tween,Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983,“Biopolymers” 22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R.Langer, et al., 1981, “J. Biomed. Mater. Res.” 15: 167-277 and R.Langer, 1982, Chem. Tech.” 12: 98-105), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121A; Epstein et al., 1985,“Proc. Natl. Acad. Sci. USA” 82: 3688-3692; Hwang et al., 1980, “Proc.Natl. Acad. Sci. USA” 77: 40304034; EP 52322A; EP 36676A; EP 88046A; EP143949A; EP 142641A; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT4 therapy.

An effective amount of a molecule of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

Further details of the invention will be apparent from the followingnon-limiting examples.

REFERENCE EXAMPLE 1 Identification of the FLS 139 Ligand

FLS139 was identified in a cDNA library prepared from human fetal livermRNA obtained from Clontech Laboratories, Inc. Palo Alto, Calif. USA,catalog no. 64018-1, following the protocol described in “InstructionManual: Superscript® Lambda System for cDNA Synthesis and λ cloning,”cat. No. 19643-014, Life Technologies, Gaithersburg, Md., USA which isherein incorporated by reference. Unless otherwise noted, all reagentswere also obtained from Life Technologies. The overall procedure can besummarized into the following steps: (1) First strand synthesis; (2)Second strand synthesis; (3) Adaptor addition; (4) Enzymatic digestion;(5) Gel isolation of cDNA; (6) Ligation into vector; and (7)Transformation.

First Strand Synthesis:

Not1 primer-adapter (Life Tech., 2 μl, 0.5 μg/μl) was added to a sterile1.5 ml microcentrifuge tube to which was added poly A+mRNA (7 μl, 5 μg).The reaction tube was heated to 70° C. for 5 minutes or time sufficientto denature the secondary structure of the mRNA. The reaction was thenchilled on ice and 5× First strand buffer (Life Tech., 4 μl), 0.1 M DTT(2 μl) and 10 mM dNTP Mix (Life Tech., 1 μl) were added and then heatedto 37° C. for 2 minutes to equilibrate the temperature. Superscript II®reverse transcriptase (Life Tech., 5 μl) was then added, the reactiontube mixed well and incubated at 37° C. for 1 hour, and terminated byplacement on ice. The final concentration of the reactants was thefollowing: 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 MM MgCl₂; 10 mM DTT;500 μM each dATP, dCTP, dGTP and dTTP; 50 μg/ml Not 1 primer-adapter; 5μg (250 μg/ml) mRNA; 50,000 U/ml Superscript II® reverse transcriptase.

Second Strand Synthesis:

While on ice, the following reagents were added to the reaction tubefrom the first strand synthesis, the reaction well mixed and allowed toreact at 16° C. for 2 hours, taking care not to allow the temperature togo above 16° C.: distilled water (93 μl); 5× Second strand buffer (30μl); dNTP mix (3 μl); 10 U/μl E. Coli DNA ligase (1 μl); 10 U/μl E. ColiDNA polymerase I (4 μl); 2 U/μl E. Coli RNase H (1 μl). 10 U T4 DNAPolymerase (2 μl) was added and the reaction continued to incubate at16° C. for another 5 minutes. The final concentration of the reactionwas the following: 25 mM Tris-HCl (pH 7.5); 100 mM KCl; 5 mM MgCl₂; 10mM (NH₄)₂SO₄; 0.15 mM G-μM each dATP, dCTP, dGTP, dTTP; 1.2 mM DTT; 65U/ml DNA ligase; 250 U/ml DNA polymerase I; 13 U/ml Rnase H. Thereaction has halted by placement on ice and by addition of 0.5 M EDTA(10 μl), then extracted through phenol:chloroform:isoamyl alcohol(25:24:1, 150 μl). The aqueous phase was removed, collected and dilutedinto 5M NaCl (15 μl) and absolute ethanol (−20° C., 400 μl) andcentrifuged for 2 minutes at 14,000×g. The supernatant was carefullyremoved from the resulting DNA pellet, the pellet resuspended in 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000×g. Thesupernatant was again removed and the pellet dried in a speedvac.

Adapter Addition

The following reagents were added to the cDNA pellet from the Secondstrand synthesis above, and the reaction was gently mixed and incubatedat 16° C. for 16 hours: distilled water (25 μl); 5× T4 DNA ligase buffer(10 μl); Sal I adapters (10 μl); T4 DNA ligase (5 μl). The finalcomposition of the reaction was the following: 50 mM Tris-HCl (pH 7.6);10 mM MgCl₂; 1 mM ATP; 5% (w/v) PEG 8000; 1 mM DTT; 200 μg/ml Sal 1adapters; 100 U/ml T4 DNA ligase. The reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 50 μl), the aqueous phaseremoved, collected and diluted into 5M NaCl (8 μl) and absolute ethanol(−20° C., 250 μl). This was then centrifuged for 20 minutes at 14,000×g,the supernatant removed and the pellet was resuspended in 0.5 ml 70%ethanol, and centrifuged again for 2 minutes at 14,000×g. Subsequently,the supernatant was removed and the resulting pellet dried in a speedvacand carried on into the next procedure.

Enzymatic Digestion:

To the cDNA prepared with the Sal 1 adapter from the previous paragraphwas added the following reagents and the mixture was incubated at 37° C.for 2 hours: DEPC-treated water (41 μl); Not 1 restriction buffer(REACT, Life Tech., 5 μl), Not 1 (4 μl). The final composition of thisreaction was the following: 50 mM Tris-HCl (pH 8.0); 10 MM MgCl₂; 100 mMMaCl; 1,200 U/ml Not 1.

Gel Isolation of cDNA:

The cDNA is size fractionated by acrylamide gel electrophoresis on a 5%acrylamide gel, and any fragments which were larger than 1 Kb, asdetermined by comparison with a molecular weight marker, were excisedfrom the gel. The cDNA was then electroeluted from the gel into 0.1×TBEbuffer (200 μl) and extracted with phenol:chloroform:isoamyl alcohol(25:24:1, 200 μl ). The aqueous phase was removed, collected andcentrifuged for 20 minutes at 14,000×g. The supernatant was removed fromthe DNA pellet which was resuspended in 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000×g. The supernatant was againdiscarded, the pellet dried in a speedvac and resuspended in distilledwater (15 μl).

Ligation of cDNA into pRK5 Vector:

The following reagents were added together and incubated at 16 ° C. for16 hours: 5× T4 ligase buffer (3 μl); pRK5, Xho1, Not1 digested vector,0.5 μg, 1 μl); cDNA prepared from previous paragraph (5 μl) anddistilled water (6 μl). Subsequently, additional distilled water (70 μl)and 10 mg/ml tRNA (0.1 μl) were added and the entire reaction wasextracted through phenol:chloroform:isoamyl alcohol (25:24:1). Theaqueous phase was removed, collected and diluted into 5M NaCl (10 μl)and absolute ethanol (−20° C., 250 μl). This was then centrifuged for 20minutes at 14,000×g, decanted, and the pellet resuspended into 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000×g. TheDNA pellet was then dried in a speedvac and eluted into distilled water(3 μl) for use in the subsequent procedure.

Transformation of Library Ligation into Bacteria:

The ligated cDNA/pRK5 vector DNA prepared previously was chilled on iceto which was added electrocompetent DH10B bacteria (Life Tech., 20 μl).The bacterial vector mixture was then electroporated as per themanufacturers recommendation. Subsequently SOC media (1 ml) was addedand the mixture was incubated at 37° C. for 30 minutes. Thetransformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (370° C.) to allow thecolonies to grow. Positive colonies were then scraped off and the DNAisolated from the bacterial pellet using standard CsCl-gradientprotocols. For example, Ausubel et al., 2.3.1.

Identification of FLS139

FLS139 can be identified in the human fetal liver library by anystandard method known in the art, including the methods reported byKlein R. D. et al. (1996), Proc. Natl Acad. Sci. 93, 7108-7113 andJacobs (U.S. Pat. No. 5,563,637 issued Jul. 16, 1996). According toKlein et al. and Jacobs, cDNAs encoding novel secreted andmembrane-bound mammalian proteins are identified by detecting theirsecretory leader sequences using the yeast invertase gene as a reportersystem. The enzyme invertase catalyzes the breakdown of sucrose toglucose and fructose as well as the breakdown of raffinose to sucroseand melibiose. The secreted form of invertase is required for theutilization of sucrose by yeast (Saccharomyces cerevisiae) so that yeastcells that are unable to produce secreted invertase grow poorly on mediacontaining sucrose as the sole carbon and energy source. Both Klein R.D., supra, and Jacobs, supra, take advantage of the known ability ofmammalian signal sequences to functionally replace the native signalsequence of yeast invertase. A mammalian cDNA library is ligated to aDNA encoding a nonsecreted yeast invertase, the ligated DNA is isolatedand transformed into yeast cells that do not contain an, invertase gene.Recombinants containing the nonsecreted yeast invertase gene ligated toa mammalian signal sequence are identified based upon their ability togrow on a medium containing only sucrose or only raffinose as the carbonsource. The mammalian signal sequences identified are then used toscreen a second, full-length cDNA library to isolate the full-lengthclones encoding the corresponding secreted proteins.

The nucleotide sequence of FLS139 in shown in FIGS. 1A-1 and 1A-2 (SEQ.ID. NO: 16), while its amino acid sequence is shown in FIGS. 1B-1 and1B-2 (SEQ. ID. NO:17). FLS139 contains a fibrinogen-like domainexhibiting a high degree of sequence homology with the two known humanligands of the TIE-2 receptor (h-TIE2L1 and h-TIE2L2). Accordingly,FLS139 has been identified as a novel member of the TIE ligand family.

A clone of FLS139 was deposited with the American Type CultureCollection (ATCC), 1081 University Boulevard, Manassa, Va. 20110-2209,on Sep. 18, 1997 under the terms of the Budapest Treaty, and has beenassigned the deposit number ATCC20928.

EXAMPLE 1 Identification of NL1

NL1 was identified by screening the GenBank database using the computerprogram BLAST (Altshul et al., Methods in Enzymology 26:460-480 (1996).The NL1 sequence shows homology with known expressed sequence tag (EST)sequences T35448, T11442, and W77823. None of the known EST sequenceshave been identified as full length sequences, or described as ligandsassociated with the TIE receptors.

Following its identification, NL1 was cloned from a human fetal lunglibrary prepared from mRNA purchased from Clontech, Inc. (Palo Alto,Calif., USA), catalog #6528-1, following the manufacturer'sinstructions. The library was screened by hybridization with syntheticoligonucleotide probes:

NL1.5-1 5′-GCTGACGAACCAAGGCAACTACAAACTCCTGGT SEQ. ID. NO: 7

NL1.3-1 5′-TGCGGCCGGACCAGTCCTCCATGGTCACCAGGAGTTTGTAG SEQ. ID. NO: 8

NL1.3-2 5′-GGTGGTGAACTGCTTGCCGTTGTGCCATGTAAA SEQ. ID. NO: 9

based on the ESTs found in the GenBank database. cDNA sequences weresequenced in their entireties.

The nucleotide and amino acid sequences of NL1 are shown in FIGS. 2A and2B (SEQ. ID. NO:1) and FIGS. 3A and 3B (SEQ. ID. NO:2), respectively.

NL1 shows 23% sequence identity with both the TIE1 and the TIE2 ligand.

A clone of NL1 and deposited with the American Type Culture Collection(ATCC), 1081 University Boulevard, Manassa, Va. 20110-2209, on Sep. 18,1997 under the terms of the Budapest Treaty, and has been assigned thedeposit number ATCC20928.

EXAMPLE 2 Identification of NL5 and NL8

An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and ESTs wereidentified that showed homology to the FLS139 protein of ReferenceExample 1. To clone NL5 and NL8, a human fetal lung library preparedfrom mRNA purchased from Clontech, Inc. (Palo Alto, Calif., USA),catalog #6528-1 was used, following the manufacturer's instructions. Thelibrary was screened by hybridization with synthetic oligonucleotideprobes:

NL5

NL5.5-1 5′ CAGGTTATCCCAGAGATTTAATGCCACCA SEQ. ID. NO: 10

NL5.3-1 5′ TTGGTGGGAGAAGTTGCCAGATCAGGTGGTGGCA SEQ. ID. NO: 11

NL5.3-2 5′ TTCACACCATAACTGCATTGGTCCA SEQ. ID. NO: 12

NL8

NL8.5-1 5′ ACGTAGTTCCAGTATGGTGTGAGCAGCAACTGGA SEQ. ID. NO: 13

NL8.3-1 5′ AGTCCAGCCTCCACCCTCCAGTTGCT SEQ. ID. NO: 14

NL8.3-2 5′ CCCCAGTCCTCCAGGAGAACCAGCA SEQ. ID. NO: 15

based on the ESTs found in the database. cDNA sequences were sequencedin their entireties. cDNA clones were sequenced. The entire nucleotideand deduced amino acid sequences of NL5 are shown in FIGS. 4A-4C and5A-5B (SEQ. ID. Nos: 3 and 4). The entire nucleotide and deduced aminoacid sequences of NL8 are shown in FIGS. 6A-6B and 7A-7B (SEQ. ID. Nos:5 and 6).

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNprogram) of the full-length sequences, NL5 shows a 24% sequence identitywith both ligand 1 and ligand 2 of the TIE2 receptor. NL8 shows a 23%sequence identity with both ligand 1 and ligand 2 of the TIE2 receptor.

The fibrinogen domains of the TIE ligand homologues NL1, NL5 and NL8 are64-74% identical. More specifically, the fibrinogen domain of NL1 is 74%identical with the fibrinogen domain of NL5 and 63% identical with thefibrinogen domain of NL8, while the fibrinogen domain of NL5 is 57%identical with the fibrinogen domain of NL8. Ligand 1 and ligand 2 ofthe TIE-2 receptor are 64% identical and 40-43% identical to NL1, NL5and NL8.

EXAMPLE 3 Northern Blot and in situ RNA Hybridization Analysis

Expression of the NL1 and NL5 mRNA in human tissues was examined byNorthern blot analysis. Human mRNA blots were hybridized to a³²P-labeled DNA probe based on the full length cDNAs; the probes weregenerated by digesting and purifying the cDNA inserts. Human fetal RNAblot MTN (Clontech) and human adult RNA blot MTN-II (Clontech) wereincubated with the DNA probes. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2Denhardt's solution; 100 mg/mL denaturedsheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42° C.The blots were washed several times in 2×SSC; 0.05% SDS for 1 hour atroom temperature, followed by a 30 minute wash in 0.1×SSC; 0.1% SDS at50° C. The blots were developed after overnight exposure byphosphorimager analysis (Fuji).

As shown in FIGS. 11 and 12, NL1 and NL5 mRNA transcripts were detected.Strong NL1 mRNA expression was detected in heart and skeletal muscletissue and in the pancreas. NL5 mRNA was strongly expressed in skeletalmuscle, and, to a lesser degree, heart, placenta and pancreas.

In situ hybridization results show that NL1 is expressed in thecartilage of developing long bones and in periosteum adjacent todifferentiating osteoblasts. Expression was also observed in connectivetissue at sites of synovial joint formation, in connective tissue septa,and in the periosteum of fetal body wall (FIGS. 8A and 8B).

In situ hybridization was performed using an optimized protocol, usingPCR-generated ³³P-labeled riboprobes. (Lu and Gillett, Cell Vision 1:169-176 (1994)). Formalin-fixed, paraffin-embedded human fetal and adulttissues were sectioned, deparaffinized, deproteinated in proteinase K(20 g/ml) for 15 minutes at 37° C., and further processed for in situhybridization as described by Lu and Gillett (1994). A [³³-P]UTP-labeled antisense riboprobe was generated from a PCR product andhybridized at 55° C. overnight. The slides were dipped in Kodak NTB2nuclear track emulsion and exposed for 4 weeks.

In situ hybridization indicated NL5 mRNA expression in adult humanbreast cancel cells over benign breast epithelium, areas of apocrinemetaplasia and sclerosing adenosis. Expression was further observed overinfiltrating breast ductal carcinoma cells. In fetal lower limb tissue,high expression was found at sites of enchondral bone formation, inosteocytes and in periosteum/perichondrium of developing bones. NL5 mRNAwas also highly expressed in osteocytes and in periosteum/periochondriumof developing bones of fetal body wall tissue. This distributionsuggests a role in bone formation and differentiation (FIGS. 9A and 9B).

In situ hybridization for NL8 showed highly organized expression patternin the developing limb, intestine and body wall, suggesting adistinctive functional role at this site, and potential involvement inangiogenesis and patterning (FIGS. 10A and 10B). This expression patternis distinct from that of NL1 and NL5.

EXAMPLE 4 Expression of NL1, NL5, and NL8 in E. coli

This example illustrates the preparation of an unglycosylated form ofthe TIE ligand homologues of the present invention in E. coli. The DNAsequence encoding a NL1, NL5 or NL8 ligand (SEQ. ID. NOs: 1, 3, and 5,respectively) is initially amplified using selected PCR primers. Theprimers should contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector. A variety ofexpression vectors may be employed. The vector will preferably encode anantibiotic resistance gene, an origin of replication, e promoter, and aribozyme binding site. An example of a suitable vector is pBR322(derived from E. coli; see Bolivar et al., Gene 2:95 (1977)) whichcontains genes for ampicillin and tetracycline resistance. The vector isdigested with restriction enzyme and dephosphorylated. The PCR amplifiedsequences are then ligated into the vector.

The ligation mixture is then used to transform a selected E. colistrain, using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis.

Selected clones can be grown overnight in liquid culture medium such as1B broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a later scale culture. The cells arethen grown to a desired optical density. An inducer, such as IPTG may beadded.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized protein can then be purified using a metal chelatingcolumn under conditions that allow tight binding of the protein.

EXAMPLE 5 Expression of NL1, NL5 and NL8 in Mammalian Cells

This example illustrates preparation of a glycosylated form of the NL1,NL5 and NL8 ligands by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the NL1, NL5 and NL8 DNA isligated into pRK5 with selected restriction enzymes to allow insertionof the NL1, NL5 and NL8 DNA using ligation methods such as described inSambrook et al., supra. The resulting vector is called pRK5-NL1, NL5 andNL8, respectively.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-NL1, NL5 and NL8 DNA is mixed with about 1 μg DNA encoding the VARNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture isadded, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mMNaPO₄, and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of NL1, NL5 and NL8 polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

In an alternative technique, NL1, NL5 and NL8 may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-NL1, NL5 andNL8 DNA is added. The cells are first concentrated from the spinnerflask by centrifugation and washed with PBS. The DNA-dextran precipitateis incubated on the cell pellet for four hours. The cells are treatedwith 20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed NL1, NL5 and NL8 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, NL1, NL5 and NL8 can be expressed in CHO cells.The pRK5-NL1, NL5 and NL8 can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of NL1, NL5 and NL8 polypeptide, the culturemedium may be replaced with serum free medium. Preferably, the culturesare incubated for about 6 days, and then the conditioned medium isharvested. The medium containing the expressed NL1, NL5 and NL8 can thenbe concentrated and purified by any selected method.

Epitope-tagged NL1, NL5 and NL8 may also be expressed in host CHO cells.NL1, NL5 and NL8 may be subcloned out of the pRK5 vector. The subcloneinsert can undergo PCR to fuse in frame with a selected epitope tag suchas a poly-his tag into a Baculovirus expression vector. The poly-histagged NL1, NL5 and NL8 insert can then be subcloned into a SV40 drivenvector containing a selection marker such as DHFR for selection ofstable clones. Finally, the CHO cells can be transfected (as describedabove) with the SV40 driven vector. Labeling may be performed, asdescribed above, to verify expression. The culture medium containing theexpressed poly-His tagged NL1, NL5 and NL8 can then be concentrated andpurified by any selected method, such as by Ni²′-chelate affinitychromatography.

EXAMPLE 6 Expression of NL1, NL5 and NL8 in Yeast

First, yeast expression vectors are constructed for intracellularproduction or secretion of NL1, NL5 and NL8 from the ADH2/GAPDHpromoter. DNA encoding NL1, NL5 and NL8, a selected signal peptide andthe promoter is inserted into suitable restriction enzyme sites in theselected plasmid to direct intracellular expression of NL1, NL5 and NL8.For secretion, DNA encoding NL1, NL5 and NL8 can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter,the yeast alpha-factor secretory signal/leader sequence, and linkersequences (if needed) for expression of NL1, NL5 and NL8.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant NL1, NL5 and NL8 can subsequently be isolated and purifiedby removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing NL1, NL5 and NL8 mayfurther be purified using selected column chromatography resins.

EXAMPLE 7 Expression of NL1, NL2 and NL8 in Baculovirus

The following method describes recombinant expression of NL1, NL5 andNL8 in Baculovirus.

The NL1, NL5 and NL8 is fused upstream of an epitope tag contained witha baculovirus expression vector. Such epitope tags include poly-his tagsand immunoglobulin tags (like Fc regions of IgG). A variety of plasmidsmay be employed, including plasmids derived from commercially availableplasmids such as pVL1393 (Novagen). Briefly, the NL1, NL5 and NL8 or thedesired portion of the NL1, NL5 and NL8 (such as the sequence encodingthe extracellular domain of a transmembrane protein) is amplified by PCRwith primers complementary to the 5′ and 3′ regions. The 5′ primer mayincorporate flanking (selected) restriction enzyme sites. The product isthen digested with those selected restriction enzymes and subcloned intothe expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged NL1, NL5 and NL8 can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged NL1, NL5 and NL8 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) NL1, NL5and NL8 can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

EXAMPLE 8 Preparation of Antibodies that bind NL1, NL2 and NL8

This example illustrates preparation of monoclonal antibodies which canspecifically bind NL1, NL2 and NL8.

Techniques for producing the monoclonal antibodies are known in the artand are described, for example, in Goding, supra. Immunogens that may beemployed include purified ligands of the present invention, fusionproteins containing such ligands, and cells expressing recombinantligands on the cell surface. Selection of the immunogen can be made bythe skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms.

Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind food pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice might also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing ELISAassays to detect the antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of the given ligand. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe antigen. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against the TIE ligand homologues hereinis well within the skill in the art.

The positive hybridoma cells can be injected intraperitoneal intosyngeneic Balb/c mice to produce ascites containing the anti-TIE-ligandhomologues monoclonal antibodies. Alternatively, the hybridoma cells canbe grown in tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Deposit of Material

As noted before, the following materials have been deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassa,Va. 20110-2209, USA (ATCC):

Material ATCC Dep. No. Deposit Date NL1-DNA22779-1130 ATCC209280 Sep.18, 1997 NL5-DNA28497-1130 ATCC209279 Sep. 18, 1997 NL8-DNA 23339-1130ATCC209282 Sep. 18, 1997

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of the deposit. The deposit will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC §122 and Commissioner's rulespursuant thereto (including 37 C.F.R. §1.14 with particular reference to886 OG 683).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die to be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The present specification is considered to be sufficient to enable oneskilled in the art to practice the invention. The present invention isnot to be limited in scope by the construct deposited, since thedeposited embodiment is intended as a single illustration of certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of the invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription is inadequate to enable the practice of any aspect of theinvention, including the best more thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustrations that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

17 2290 base pairs Nucleic Acid Single Linear 1 GGCTGAGGGG AGGCCCGGAGCCTTTCTGGG GCCTGGGGGA TCCTCTTGCA 50 CTGGTGGGTG GAGAGAAGCG CCTGCAGCCAACCAGGGTCA GGCTGTGCTC 100 ACAGTTTCCT CTGGCGGCAT GTAAAGGCTC CACAAAGGAGTTGGGAGTTC 150 AAATGAGGCT GCTGCGGACG GCCTGAGGAT GGACCCCAAG CCCTGGACCT200 GCCGAGCGTG GCACTGAGGC AGCGGCTGAC GCTACTGTGA GGGAAAGAAG 250GTTGTGAGCA GCCCCGCAGG ACCCCTGGCC AGCCCTGGCC CCAGCCTCTG 300 CCGGAGCCCTCTGTGGAGGC AGAGCCAGTG GAGCCCAGTG AGGCAGGGCT 350 GCTTGGCAGC CACCGGCCTGCAACTCAGGA ACCCCTCCAG AGGCCATGGA 400 CAGGCTGCCC CGCTGACGGC CAGGGTGAAGCATGTGAGGA GCCGCCCCGG 450 AGCCAAGCAG GAGGGAAGAG GCTTTCATAG ATTCTATTCACAAAGAATAA 500 CCACCATTTT GCAAGGACCA TGAGGCCACT GTGCGTGACA TGCTGGTGGC550 TCGGACTGCT GGCTGCCATG GGAGCTGTTG CAGGCCAGGA GGACGGTTTT 600GAGGGCACTG AGGAGGGCTC GCCAAGAGAG TTCATTTACC TAAACAGGTA 650 CAAGCGGGCGGGCGAGTCCC AGGACAAGTG CACCTACACC TTCATTGTGC 700 CCCAGCAGCG GGTCACGGGTGCCATCTGCG TCAACTCCAA GGAGCCTGAG 750 GTGCTTCTGG AGAACCGAGT GCATAAGCAGGAGCTAGAGC TGCTCAACAA 800 TGAGCTGCTC AAGCAGAAGC GGCAGATCGA GACGCTGCAGCAGCTGGTGG 850 AGGTGGACGG CGGCATTGTG AGCGAGGTGA AGCTGCTGCG CAAGGAGAGC900 CGCAACATGA ACTCGCGGGT CACGCAGCTC TACATGCAGC TCCTGCACGA 950GATCATCCGC AAGCGGGACA ACGCGTTGGA GCTCTCCCAG CTGGAGAACA 1000 GGATCCTGAACCAGACAGCC GACATGCTGC AGCTGGCCAG CAAGTACAAG 1050 GACCTGGAGC ACAAGTACCAGCACCTGGCC ACACTGGCCC ACAACCAATC 1100 AGAGATCATC GCGCAGCTTG AGGAGCACTGCCAGAGGGTG CCCTCGGCCA 1150 GGCCCGTCCC CCAGCCACCC CCCGCTGCCC CGCCCCGGGTCTACCAACCA 1200 CCCACCTACA ACCGCATCAT CAACCAGATC TCTACCAACG AGATCCAGAG1250 TGACCAGAAC CTGAAGGTGC TGCCACCCCC TCTGCCCACT ATGCCCACTC 1300TCACCAGCCT CCCATCTTCC ACCGACAAGC CGTCGGGCCC ATGGAGAGAC 1350 TGCCTGCAGGCCCTGGAGGA TGGCCACGAC ACCAGCTCCA TCTACCTGGT 1400 GAAGCCGGAG AACACCAACCGCCTCATGCA GGTGTGGTGC GACCAGAGAC 1450 ACGACCCCGG GGGCTGGACC GTCATCCAGAGACGCCTGGA TGGCTCTGTT 1500 AACTTCTTCA GGAACTGGGA GACGTACAAG CAAGGGTTTGGGAACATTGA 1550 CGGCGAATAC TGGCTGGGCC TGGAGAACAT TTACTGGCTG ACGAACCAAG1600 GCAACTACAA ACTCCTGGTG ACCATGGAGG ACTGGTCCGG CCGCAAAGTC 1650TTTGCAGAAT ACGCCAGTTT CCGCCTGGAA CCTGAGAGCG AGTATTATAA 1700 GCTGCGGCTGGGGCGCTACC ATGGCAATGC GGGTGACTCC TTTACATGGC 1750 ACAACGGCAA GCAGTTCACCACCCTGGACA GAGATCATGA TGTCTACACA 1800 GGAAACTGTG CCCACTACCA GAAGGGAGGCTGGTGGTATA ACGCCTGTGC 1850 CCACTCCAAC CTCAACGGGG TCTGGTACCG CGGGGGCCATTACCGGAGCC 1900 GCTACCAGGA CGGAGTCTAC TGGGCTGAGT TCCGAGGAGG CTCTTACTCA1950 CTCAAGAAAG TGGTGATGAT GATCCGACCG AACCCCAACA CCTTCCACTA 2000AGCCAGCTCC CCCTCCTGAC CTCTCGTGGC CATTGCCAGG AGCCCACCCT 2050 GGTCACGCTGGCCACAGCAC AAAGAACAAC TCCTCACCAG TTCATCCTGA 2100 GGCTGGGAGG ACCGGGATGCTGGATTCTGT TTTCCGAAGT CACTGCAGCG 2150 GATGATGGAA CTGAATCGAT ACGGTGTTTTCTGTCCCTCC TACTTTCCTT 2200 CACACCAGAC AGCCCCTCAT GTCTCCAGGA CAGGACAGGACTACAGACAA 2250 CTCTTTCTTT AAATAAATTA AGTCTCTACA ATAAAAAAAA 2290 493amino acids Amino Acid Linear 2 Met Arg Pro Leu Cys Val Thr Cys Trp TrpLeu Gly Leu Leu Ala 1 5 10 15 Ala Met Gly Ala Val Ala Gly Gln Glu AspGly Phe Glu Gly Thr 20 25 30 Glu Glu Gly Ser Pro Arg Glu Phe Ile Tyr LeuAsn Arg Tyr Lys 35 40 45 Arg Ala Gly Glu Ser Gln Asp Lys Cys Thr Tyr ThrPhe Ile Val 50 55 60 Pro Gln Gln Arg Val Thr Gly Ala Ile Cys Val Asn SerLys Glu 65 70 75 Pro Glu Val Leu Leu Glu Asn Arg Val His Lys Gln Glu LeuGlu 80 85 90 Leu Leu Asn Asn Glu Leu Leu Lys Gln Lys Arg Gln Ile Glu Thr95 100 105 Leu Gln Gln Leu Val Glu Val Asp Gly Gly Ile Val Ser Glu Val110 115 120 Lys Leu Leu Arg Lys Glu Ser Arg Asn Met Asn Ser Arg Val Thr125 130 135 Gln Leu Tyr Met Gln Leu Leu His Glu Ile Ile Arg Lys Arg Asp140 145 150 Asn Ala Leu Glu Leu Ser Gln Leu Glu Asn Arg Ile Leu Asn Gln155 160 165 Thr Ala Asp Met Leu Gln Leu Ala Ser Lys Tyr Lys Asp Leu Glu170 175 180 His Lys Tyr Gln His Leu Ala Thr Leu Ala His Asn Gln Ser Glu185 190 195 Ile Ile Ala Gln Leu Glu Glu His Cys Gln Arg Val Pro Ser Ala200 205 210 Arg Pro Val Pro Gln Pro Pro Pro Ala Ala Pro Pro Arg Val Tyr215 220 225 Gln Pro Pro Thr Tyr Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn230 235 240 Glu Ile Gln Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu245 250 255 Pro Thr Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys260 265 270 Pro Ser Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp Gly275 280 285 His Asp Thr Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn Thr Asn290 295 300 Arg Leu Met Gln Val Trp Cys Asp Gln Arg His Asp Pro Gly Gly305 310 315 Trp Thr Val Ile Gln Arg Arg Leu Asp Gly Ser Val Asn Phe Phe320 325 330 Arg Asn Trp Glu Thr Tyr Lys Gln Gly Phe Gly Asn Ile Asp Gly335 340 345 Glu Tyr Trp Leu Gly Leu Glu Asn Ile Tyr Trp Leu Thr Asn Gln350 355 360 Gly Asn Tyr Lys Leu Leu Val Thr Met Glu Asp Trp Ser Gly Arg365 370 375 Lys Val Phe Ala Glu Tyr Ala Ser Phe Arg Leu Glu Pro Glu Ser380 385 390 Glu Tyr Tyr Lys Leu Arg Leu Gly Arg Tyr His Gly Asn Ala Gly395 400 405 Asp Ser Phe Thr Trp His Asn Gly Lys Gln Phe Thr Thr Leu Asp410 415 420 Arg Asp His Asp Val Tyr Thr Gly Asn Cys Ala His Tyr Gln Lys425 430 435 Gly Gly Trp Trp Tyr Asn Ala Cys Ala His Ser Asn Leu Asn Gly440 445 450 Val Trp Tyr Arg Gly Gly His Tyr Arg Ser Arg Tyr Gln Asp Gly455 460 465 Val Tyr Trp Ala Glu Phe Arg Gly Gly Ser Tyr Ser Leu Lys Lys470 475 480 Val Val Met Met Ile Arg Pro Asn Pro Asn Thr Phe His 485 490493 3355 base pairs Nucleic Acid Single Linear 3 GCAGCTGGTT ACTGCATTTCTCCATGTGGC AGACAGAGCA AAGCCACAAC 50 GCTTTCTCTG CTGGATTAAA GACGGCCCACAGACCAGAAC TTCCACTATA 100 CTACTTAAAA TTACATAGGT GGCTTGTCAA ATTCAATTGATTAGTATTGT 150 AAAAGGAAAA AGAAGTTCCT TCTTACAGCT TGGATTCAAC GGTCCAAAAC200 AAAAATGCAG CTGCCATTAA AGTCTCAGAT GAACAAACTT CTACACTGAT 250TTTTAAAATC AAGAATAAGG GCAGCAAGTT TCTGGATTCA CTGAATCAAC 300 AGACACAAAAAGCTGGCAAT ATAGCAACTA TGAAGAGAAA AGCTACTAAT 350 AAAATTAACC CAACGCATAGAAGACTTTTT TTTCTCTTCT AAAAACAACT 400 AAGTAAAGAC TTAAATTTAA ACACATCATTTTACAACCTC ATTTCAAAAT 450 GAAGACTTTT ACCTGGACCC TAGGTGTGCT ATTCTTCCTACTAGTGGACA 500 CTGGACATTG CAGAGGTGGA CAATTCAAAA TTAAAAAAAT AAACCAGAGA550 AGATACCCTC GTGCCACAGA TGGTAAAGAG GAAGCAAAGA AATGTGCATA 600CACATTCCTG GTACCTGAAC AAAGAATAAC AGGGCCAATC TGTGTCAACA 650 CCAAGGGGCAAGATGCAAGT ACCATTAAAG ACATGATCAC CAGGATGGAC 700 CTTGAAAACC TGAAGGATGTGCTCTCCAGG CAGAAGCGGG AGATAGATGT 750 TCTGCAACTG GTGGTGGATG TAGATGGAAACATTGTGAAT GAGGTAAAGC 800 TGCTGAGAAA GGAAAGCCGT AACATGAACT CTCGTGTTACTCAACTCTAT 850 ATGCAATTAT TACATGAGAT TATCCGTAAG AGGGATAATT CACTTGAACT900 TTCCCAACTG GAAAACAAAA TCCTCAATGT CACCACAGAA ATGTTGAAGA 950TGGCAACAAG ATACAGGGAA CTAGAGGTGA AATACGCTTC CTTGACTGAT 1000 CTTGTCAATAACCAATCTGT GATGATCACT TTGTTGGAAG AACAGTGCTT 1050 GAGGATATTT TCCCGACAAGACACCCATGT GTCTCCCCCA CTTGTCCAGG 1100 TGGTGCCACA ACATATTCCT AACAGCCAACAGTATACTCC TGGTCTGCTG 1150 GGAGGTAACG AGATTCAGAG GGATCCAGGT TATCCCAGAGATTTAATGCC 1200 ACCACCTGAT CTGGCAACTT CTCCCACCAA AAGCCCTTTC AAGATACCAC1250 CGGTAACTTT CATCAATGAA GGACCATTCA AAGACTGTCA GCAAGCAAAA 1300GAAGCTGGGC ATTCGGTCAG TGGGATTTAT ATGATTAAAC CTGAAAACAG 1350 CAATGGACCAATGCAGTTAT GGTGTGAAAA CAGTTTGGAC CCTGGGGGTT 1400 GGACTGTTAT TCAGAAAAGAACAGACGGCT CTGTCAACTT CTTCAGAAAT 1450 TGGGAAAATT ATAAGAAAGG GTTTGGAAACATTGACGGAG AATACTGGCT 1500 TGGACTGGAA AATATCTATA TGCTTAGCAA TCAAGATAATTACAAGTTAT 1550 TGATTGAATT AGAAGACTGG AGTGATAAAA AAGTCTATGC AGAATACAGC1600 AGCTTTCGTC TGGAACCTGA AAGTGAATTC TATAGACTGC GCCTGGGAAC 1650TTACCAGGGA AATGCAGGGG ATTCTATGAT GTGGCATAAT GGTAAACAAT 1700 TCACCACACTGGACAGAGAT AAAGATATGT ATGCAGGAAA CTGCGCCCAC 1750 TTTCATAAAG GAGGCTGGTGGTACAATGCC TGTGCACATT CTAACCTAAA 1800 TGGAGTATGG TACAGAGGAG GCCATTACAGAAGCAAGCAC CAAGATGGAA 1850 TTTTCTGGGC CGAATACAGA GGCGGGTCAT ACTCCTTAAGAGCAGTTCAG 1900 ATGATGATCA AGCCTATTGA CTGAAGAGAG ACACTCGCCA ATTTAAATGA1950 CACAGAACTT TGTACTTTTC AGCTCTTAAA AATGTAAATG TTACATGTAT 2000ATTACTTGGC ACAATTTATT TCTACACAGA AAGTTTTTAA AATGAATTTT 2050 ACCGTAACTATAAAAGGGAA CCTATAAATG TAGTTTCATC TGTCGTCAAT 2100 TACTGCAGAA AATTATGTGTATCCACAACC TAGTTATTTT AAAAATTATG 2150 TTGACTAAAT ACAAAGTTTG TTTTCTAAAATGTAAATATT TGCCACAATG 2200 TAAAGCAAAT CTTAGCTATA TTTTAAATCA TAAATAACATGTTCAAGATA 2250 CTTAACAATT TATTTAAAAT CTAAGATTGC TCTAACGTCT AGTGAAAAAA2300 ATATTTTTTA AATTTCAGCC AAATAATGCA TTTTATTTTA TAAAAATACA 2350GACAGAAAAT TAGGGAGAAA CTTCTAGTTT TGCCAATAGA AAATGTTCTT 2400 CCATTGAATAAAAGTTATTT CAAATTGAAT TTGTGCCTTT CACACGTAAT 2450 GATTAAATCT GAATTCTTAATAATATATCC TATGCTGATT TTCCCAAAAC 2500 ATGACCCATA GTATTAAATA CATATCATTTTTAAAAATAA AAAAAAACCC 2550 AAAAATAATG CATGCATAAT TTAAATGGTC AATTTATAAAGACAAATCTA 2600 TGAATGAATT TTTCAGTGTT ATCTTCATAT GATATGCTGA ACACCAAAAT2650 CTCCAGAAAT GCATTTTATG TAGTTCTAAA ATCAGCAAAA TATTGGTATT 2700ACAAAAATGC AGAATATTTA GTGTGCTACA GATCTGAATT ATAGTTCTAA 2750 TTTATTATTACTTTTTTTCT AATTTACTGA TCTTACTACT ACAAAGAAAA 2800 AAAAACCCAA CCCATCTGCAATTCAAATCA GAAAGTTTGG ACAGCTTTAC 2850 AAGTATTAGT GCATGCTCAG AACAGGTGGGACTAAAACAA ACTCAAGGAA 2900 CTGTTGGCTG TTTTCCCGAT ACTGAGAATT CAACAGCTCCAGAGCAGAAG 2950 CCACAGGGGC ATAGCTTAGT CCAAACTGCT AATTTCATTT TACAGTGTAT3000 GTAACGCTTA GTCTCACAGT GTCTTTAACT CATCTTTGCA ATCAACAACT 3050TTACTAGTGA CTTTCTGGAA CAATTTCCTT TCAGGAATAC ATATTCACTG 3100 CTTAGAGGTGACCTTGCCTT AATATATTTG TGAAGTTAAA ATTTTAAAGA 3150 TAGCTCATGA AACTTTTGCTTAAGCAAAAA GAAAACCTCG AATTGAAATG 3200 TGTGAGGCAA ACTATGCATG GGAATAGCTTAATGTGAAGA TAATCATTTG 3250 GACAACTCAA ATCCATCAAC ATGACCAATG TTTTTCATCTGCCACATCTC 3300 AAAATAAAAC TTCTGGTGAA ACAAATTAAA CAAAATATCC AAACCTCAAA3350 AAAAA 3355 491 amino acids Amino Acid Linear 4 Met Lys Thr Phe ThrTrp Thr Leu Gly Val Leu Phe Phe Leu Leu 1 5 10 15 Val Asp Thr Gly HisCys Arg Gly Gly Gln Phe Lys Ile Lys Lys 20 25 30 Ile Asn Gln Arg Arg TyrPro Arg Ala Thr Asp Gly Lys Glu Glu 35 40 45 Ala Lys Lys Cys Ala Tyr ThrPhe Leu Val Pro Glu Gln Arg Ile 50 55 60 Thr Gly Pro Ile Cys Val Asn ThrLys Gly Gln Asp Ala Ser Thr 65 70 75 Ile Lys Asp Met Ile Thr Arg Met AspLeu Glu Asn Leu Lys Asp 80 85 90 Val Leu Ser Arg Gln Lys Arg Glu Ile AspVal Leu Gln Leu Val 95 100 105 Val Asp Val Asp Gly Asn Ile Val Asn GluVal Lys Leu Leu Arg 110 115 120 Lys Glu Ser Arg Asn Met Asn Ser Arg ValThr Gln Leu Tyr Met 125 130 135 Gln Leu Leu His Glu Ile Ile Arg Lys ArgAsp Asn Ser Leu Glu 140 145 150 Leu Ser Gln Leu Glu Asn Lys Ile Leu AsnVal Thr Thr Glu Met 155 160 165 Leu Lys Met Ala Thr Arg Tyr Arg Glu LeuGlu Val Lys Tyr Ala 170 175 180 Ser Leu Thr Asp Leu Val Asn Asn Gln SerVal Met Ile Thr Leu 185 190 195 Leu Glu Glu Gln Cys Leu Arg Ile Phe SerArg Gln Asp Thr His 200 205 210 Val Ser Pro Pro Leu Val Gln Val Val ProGln His Ile Pro Asn 215 220 225 Ser Gln Gln Tyr Thr Pro Gly Leu Leu GlyGly Asn Glu Ile Gln 230 235 240 Arg Asp Pro Gly Tyr Pro Arg Asp Leu MetPro Pro Pro Asp Leu 245 250 255 Ala Thr Ser Pro Thr Lys Ser Pro Phe LysIle Pro Pro Val Thr 260 265 270 Phe Ile Asn Glu Gly Pro Phe Lys Asp CysGln Gln Ala Lys Glu 275 280 285 Ala Gly His Ser Val Ser Gly Ile Tyr MetIle Lys Pro Glu Asn 290 295 300 Ser Asn Gly Pro Met Gln Leu Trp Cys GluAsn Ser Leu Asp Pro 305 310 315 Gly Gly Trp Thr Val Ile Gln Lys Arg ThrAsp Gly Ser Val Asn 320 325 330 Phe Phe Arg Asn Trp Glu Asn Tyr Lys LysGly Phe Gly Asn Ile 335 340 345 Asp Gly Glu Tyr Trp Leu Gly Leu Glu AsnIle Tyr Met Leu Ser 350 355 360 Asn Gln Asp Asn Tyr Lys Leu Leu Ile GluLeu Glu Asp Trp Ser 365 370 375 Asp Lys Lys Val Tyr Ala Glu Tyr Ser SerPhe Arg Leu Glu Pro 380 385 390 Glu Ser Glu Phe Tyr Arg Leu Arg Leu GlyThr Tyr Gln Gly Asn 395 400 405 Ala Gly Asp Ser Met Met Trp His Asn GlyLys Gln Phe Thr Thr 410 415 420 Leu Asp Arg Asp Lys Asp Met Tyr Ala GlyAsn Cys Ala His Phe 425 430 435 His Lys Gly Gly Trp Trp Tyr Asn Ala CysAla His Ser Asn Leu 440 445 450 Asn Gly Val Trp Tyr Arg Gly Gly His TyrArg Ser Lys His Gln 455 460 465 Asp Gly Ile Phe Trp Ala Glu Tyr Arg GlyGly Ser Tyr Ser Leu 470 475 480 Arg Ala Val Gln Met Met Ile Lys Pro IleAsp 485 490 491 1780 base pairs Nucleic Acid Single Linear 5 GGCTCAGAGGCCCCACTGGA CCCTCGGCTC TTCCTTGGAC TTCTTGTGTG 50 TTCTGTGAGC TTCGCTGGATTCAGGGTCTT GGGCATCAGA GGTGAGAGGG 100 TGGGAAGGTC CGCCGCGATG GGGAAGCCCTGGCTGCGTGC GCTACAGCTG 150 CTGCTCCTGC TGGGCGCGTC GTGGGCGCGG GCGGGCGCCCCGCGCTGCAC 200 CTACACCTTC GTGCTGCCCC CGCAGAAGTT CACGGGCGCT GTGTGCTGGA250 GCGGCCCCGC ATCCACGCGG GCGACGCCCG AGGCCGCCAA CGCCAGCGAG 300CTGGCGGCGC TGCGCATGCG CGTCGGCCGC CACGAGGAGC TGTTACGCGA 350 GCTGCAGAGGCTGGCGGCGG CCGACGGCGC CGTGGCCGGC GAGGTGCGCG 400 CGCTGCGCAA GGAGAGCCGCGGCCTGAGCG CGCGCCTGGG CCAGTTGCGC 450 GCGCAGCTGC AGCACGAGGC GGGGCCCGGGGCGGGCCCGG GGGCGGATCT 500 GGGGGCGGAG CCTGCCGCGG CGCTGGCGCT GCTCGGGGAGCGCGTGCTCA 550 ACGCGTCCGC CGAGGCTCAG CGCGCAGCCG CCCGGTTCCA CCAGCTGGAC600 GTCAAGTTCC GCGAGCTGGC GCAGCTCGTC ACCCAGCAGA GCAGTCTCAT 650CGCCCGCCTG GAGCGCCTGT GCCCGGGAGG CGCGGGCGGG CAGCAGCAGG 700 TCCTGCCGCCACCCCCACTG GTGCCTGTGG TTCCGGTCCG TCTTGTGGGT 750 AGCACCAGTG ACACCAGTAGGATGCTGGAC CCAGCCCCAG AGCCCCAGAG 800 AGACCAGACC CAGAGACAGC AGGAGCCCATGGCTTCTCCC ATGCCTGCAG 850 GTCACCCTGC GGTCCCCACC AAGCCTGTGG GCCCGTGGCAGGATTGTGCA 900 GAGGCCCGCC AGGCAGGCCA TGAACAGAGT GGAGTGTATG AACTGCGAGT950 GGGCCGTCAC GTAGTGTCAG TATGGTGTGA GCAGCAACTG GAGGGTGGAG 1000GCTGGACTGT GATCCAGCGG AGGCAAGATG GTTCAGTCAA CTTCTTCACT 1050 ACCTGGCAGCACTATAAGGC GGGCTTTGGG CGGCCAGACG GAGAATACTG 1100 GCTGGGCCTT GAACCCGTGTATCAGCTGAC CAGCCGTGGG GACCATGAGC 1150 TGCTGGTTCT CCTGGAGGAC TGGGGGGGCCGTGGAGCACG TGCCCACTAT 1200 GATGGCTTCT CCCTGGAACC CGAGAGCGAC CACTACCGCCTGCGGCTTGG 1250 CCAGTACCAT GGTGATGCTG GAGACTCTCT TTCCTGGCAC AATGACAAGC1300 CCTTCAGCAC CGTGGATAGG GACCGAGACT CCTATTCTGG TAACTGTGCC 1350CTGTACCAGC GGGGAGGCTG GTGGTACCAT GCCTGTGCCC ACTCCAACCT 1400 CAACGGTGTGTGGCACCACG GCGGCCACTA CCGAAGCCGC TACCAGGATG 1450 GTGTCTACTG GGCTGAGTTTCGTGGTGGGG CATATTCTCT CAGGAAGGCC 1500 GCCATGCTCA TTCGGCCCCT GAAGCTGTGACTCTGTGTTC CTCTGTCCCC 1550 TAGGCCCTAG AGGACATTGG TCAGCAGGAG CCCAAGTTGTTCTGGCCACA 1600 CCTTCTTTGT GGCTCAGTGC CAATGTGTCC CACAGAACTT CCCACTGTGG1650 ATCTGTGACC CTGGGCGCTG AAAATGGGAC CCAGGAATCC CCCCCGTCAA 1700TATCTTGGCC TCAGATGGCT CCCCAAGGTC ATTCATATCT CGGTTTGAGC 1750 TCATATCTTATAATAACACA AAGTAGCCAC 1780 470 amino acids Amino Acid Linear 6 Met GlyLys Pro Trp Leu Arg Ala Leu Gln Leu Leu Leu Leu Leu 1 5 10 15 Gly AlaSer Trp Ala Arg Ala Gly Ala Pro Arg Cys Thr Tyr Thr 20 25 30 Phe Val LeuPro Pro Gln Lys Phe Thr Gly Ala Val Cys Trp Ser 35 40 45 Gly Pro Ala SerThr Arg Ala Thr Pro Glu Ala Ala Asn Ala Ser 50 55 60 Glu Leu Ala Ala LeuArg Met Arg Val Gly Arg His Glu Glu Leu 65 70 75 Leu Arg Glu Leu Gln ArgLeu Ala Ala Ala Asp Gly Ala Val Ala 80 85 90 Gly Glu Val Arg Ala Leu ArgLys Glu Ser Arg Gly Leu Ser Ala 95 100 105 Arg Leu Gly Gln Leu Arg AlaGln Leu Gln His Glu Ala Gly Pro 110 115 120 Gly Ala Gly Pro Gly Ala AspLeu Gly Ala Glu Pro Ala Ala Ala 125 130 135 Leu Ala Leu Leu Gly Glu ArgVal Leu Asn Ala Ser Ala Glu Ala 140 145 150 Gln Arg Ala Ala Ala Arg PheHis Gln Leu Asp Val Lys Phe Arg 155 160 165 Glu Leu Ala Gln Leu Val ThrGln Gln Ser Ser Leu Ile Ala Arg 170 175 180 Leu Glu Arg Leu Cys Pro GlyGly Ala Gly Gly Gln Gln Gln Val 185 190 195 Leu Pro Pro Pro Pro Leu ValPro Val Val Pro Val Arg Leu Val 200 205 210 Gly Ser Thr Ser Asp Thr SerArg Met Leu Asp Pro Ala Pro Glu 215 220 225 Pro Gln Arg Asp Gln Thr GlnArg Gln Gln Glu Pro Met Ala Ser 230 235 240 Pro Met Pro Ala Gly His ProAla Val Pro Thr Lys Pro Val Gly 245 250 255 Pro Trp Gln Asp Cys Ala GluAla Arg Gln Ala Gly His Glu Gln 260 265 270 Ser Gly Val Tyr Glu Leu ArgVal Gly Arg His Val Val Ser Val 275 280 285 Trp Cys Glu Gln Gln Leu GluGly Gly Gly Trp Thr Val Ile Gln 290 295 300 Arg Arg Gln Asp Gly Ser ValAsn Phe Phe Thr Thr Trp Gln His 305 310 315 Tyr Lys Ala Gly Phe Gly ArgPro Asp Gly Glu Tyr Trp Leu Gly 320 325 330 Leu Glu Pro Val Tyr Gln LeuThr Ser Arg Gly Asp His Glu Leu 335 340 345 Leu Val Leu Leu Glu Asp TrpGly Gly Arg Gly Ala Arg Ala His 350 355 360 Tyr Asp Gly Phe Ser Leu GluPro Glu Ser Asp His Tyr Arg Leu 365 370 375 Arg Leu Gly Gln Tyr His GlyAsp Ala Gly Asp Ser Leu Ser Trp 380 385 390 His Asn Asp Lys Pro Phe SerThr Val Asp Arg Asp Arg Asp Ser 395 400 405 Tyr Ser Gly Asn Cys Ala LeuTyr Gln Arg Gly Gly Trp Trp Tyr 410 415 420 His Ala Cys Ala His Ser AsnLeu Asn Gly Val Trp His His Gly 425 430 435 Gly His Tyr Arg Ser Arg TyrGln Asp Gly Val Tyr Trp Ala Glu 440 445 450 Phe Arg Gly Gly Ala Tyr SerLeu Arg Lys Ala Ala Met Leu Ile 455 460 465 Arg Pro Leu Lys Leu 470 33base pairs Nucleic Acid Single Linear 7 GCTGACGAAC CAAGGCAACT ACAAACTCCTGGT 33 41 base pairs Nucleic Acid Single Linear 8 TGCGGCCGGA CCAGTCCTCCATGGTCACCA GGAGTTTGTA G 41 33 base pairs Nucleic Acid Single Linear 9GGTGGTGAAC TGCTTGCCGT TGTGCCATGT AAA 33 29 base pairs Nucleic AcidSingle Linear 10 CAGGTTATCC CAGAGATTTA ATGCCACCA 29 34 base pairsNucleic Acid Single Linear 11 TTGGTGGGAG AAGTTGCCAG ATCAGGTGGT GGCA 3425 base pairs Nucleic Acid Single Linear 12 TTCACACCAT AACTGCATTG GTCCA25 34 base pairs Nucleic Acid Single Linear 13 ACGTAGTTCC AGTATGGTGTGAGCAGCAAC TGGA 34 26 base pairs Nucleic Acid Single Linear 14AGTCCAGCCT CCACCCTCCA GTTGCT 26 25 base pairs Nucleic Acid Single Linear15 CCCCAGTCCT CCAGGAGAAC CAGCA 25 2042 base pairs Nucleic Acid SingleLinear 16 GCGGACGCGT GGGTGAAATT GAAAATCAAG ATAAAAATGT TCACAATTAA 50GCTCCTTCTT TTTATTGTTC CTCTAGTTAT TTCCTCCAGA ATTGATCAAG 100 ACAATTCATCATTTGATTCT CTATCTCCAG AGCCAAAATC AAGATTTGCT 150 ATGTTAGACG ATGTAAAAATTTTAGCCAAT GGCCTCCTTC AGTTGGGACA 200 TGGTCTTAAA GACTTTGTCC ATAAGACGAAGGGCCAAATT AATGACATAT 250 TTCAAAAACT CAACATATTT GATCAGTCTT TTTATGATCTATCGCTGCAA 300 ACCAGTGAAA TCAAAGAAGA AGAAAAGGAA CTGAGAAGAA CTACATATAA350 ACTACAAGTC AAAAATGAAG AGGTAAAGAA TATGTCACTT GAACTCAACT 400CAAAACTTGA AAGCCTCCTA GAAGAAAAAA TTCTACTTCA ACAAAAAGTG 450 AAATATTTAGAAGAGCAACT AACTAACTTA ATTCAAAATC AACCTGAAAC 500 TCCAGAACAC CCAGAAGTAACTTCACTTAA AACTTTTGTA GAAAAACAAG 550 ATAATAGCAT CAAAGACCTT CTCCAGACCGTGGAAGACCA ATATAAACAA 600 TTAAACCAAC AGCATAGTCA AATAAAAGAA ATAGAAAATCAGCTCAGAAG 650 GACTAGTATT CAAGAACCCA CAGAAATTTC TCTATCTTCC AAGCCAAGAG700 CACCAAGAAC TACTCCCTTT CTTCAGTTGA ATGAAATAAG AAATGTAAAA 750CATGATGGCA TTCCTGCTGA ATGTACCACC ATTTATAACA GAGGTGAACA 800 TACAAGTGGCATGTATGCCA TCAGACCCAG CAACTCTCAA GTTTTTCATG 850 TCTACTGTGA TGTTATATCAGGTAGTCCAT GGACATTAAT TCAACATCGA 900 ATAGATGGAT CACAAAACTT CAATGAAACGTGGGAGAACT ACAAATATGG 950 TTTTGGGAGG CTTGATGGAG AATTTTGGTT GGGCCTAGAGAAGATATACT 1000 CCATAGTGAA GCAATCTAAT TATGTTTTAC GAATTGAGTT GGAAGACTGG1050 AAAGACAACA AACATTATAT TGAATATTCT TTTTACTTGG GAAATCACGA 1100AACCAACTAT ACGCTACATC TAGTTGCGAT TACTGGCAAT GTCCCCAATG 1150 CAATCCCGGAAAACAAAGAT TTGGTGTTTT CTACTTGGGA TCACAAAGCA 1200 AAAGGACACT TCAACTGTCCAGAGGGTTAT TCAGGAGGCT GGTGGTGGCA 1250 TGATGAGTGT GGAGAAAACA ACCTAAATGGTAAATATAAC AAACCAAGAG 1300 CAAAATCTAA GCCAGAGAGG AGAAGAGGAT TATCTTGGAAGTCTCAAAAT 1350 GGAAGGTTAT ACTCTATAAA ATCAACCAAA ATGTTGATCC ATCCAACAGA1400 TTCAGAAAGC TTTGAATGAA CTGAGGCAAT TTAAAGGCAT ATTTAACCAT 1450TAACTCATTC CAAGTTAATG TGGTCTAATA ATCTGGTATA AATCCTTAAG 1500 AGAAAGCTTGAGAAATAGAT TTTTTTTATC TTAAAGTCAC TGTCTATTTA 1550 AGATTAAACA TACAATCACATAACCTTAAA GAATACCGTT TACATTTCTC 1600 AATCAAAATT CTTATAATAC TATTTGTTTTAAATTTTGTG ATGTGGGAAT 1650 CAATTTTAGA TGGTCACAAT CTAGATTATA ATCAATAGGTGAACTTATTA 1700 AATAACTTTT CTAAATAAAA AATTTAGAGA CTTTTATTTT AAAAGGCATC1750 ATATGAGCTA ATATCACAAC TTTCCCAGTT TAAAAAACTA GTACTCTTGT 1800TAAAACTCTA AACTTGACTA AATACAGAGG ACTGGTAATT GTACAGTTCT 1850 TAAATGTTGTAGTATTAATT TCAAAACTAA AAATCGTCAG CACAGAGTAT 1900 GTGTAAAAAT CTGTAATACAAATTTTTAAA CTGATGCTTC ATTTTGCTAC 1950 AAAATAATTT GGAGTAAATG TTTGATATGATTTATTTATG AAACCTAATG 2000 AAGCAGAATT AAATACTGTA TTAAAATAAG TTCGCTGTCTTT 2042 460 amino acids Amino Acid Linear 17 Met Phe Thr Ile Lys Leu LeuLeu Phe Ile Val Pro Leu Val Ile 1 5 10 15 Ser Ser Arg Ile Asp Gln AspAsn Ser Ser Phe Asp Ser Leu Ser 20 25 30 Pro Glu Pro Lys Ser Arg Phe AlaMet Leu Asp Asp Val Lys Ile 35 40 45 Leu Ala Asn Gly Leu Leu Gln Leu GlyHis Gly Leu Lys Asp Phe 50 55 60 Val His Lys Thr Lys Gly Gln Ile Asn AspIle Phe Gln Lys Leu 65 70 75 Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu SerLeu Gln Thr Ser 80 85 90 Glu Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg ThrThr Tyr Lys 95 100 105 Leu Gln Val Lys Asn Glu Glu Val Lys Asn Met SerLeu Glu Leu 110 115 120 Asn Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys IleLeu Leu Gln 125 130 135 Gln Lys Val Lys Tyr Leu Glu Glu Gln Leu Thr AsnLeu Ile Gln 140 145 150 Asn Gln Pro Glu Thr Pro Glu His Pro Glu Val ThrSer Leu Lys 155 160 165 Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys AspLeu Leu Gln 170 175 180 Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln GlnHis Ser Gln 185 190 195 Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr SerIle Gln Glu 200 205 210 Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg AlaPro Arg Thr 215 220 225 Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn ValLys His Asp 230 235 240 Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn ArgGly Glu His 245 250 255 Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn SerGln Val Phe 260 265 270 His Val Tyr Cys Asp Val Ile Ser Gly Ser Pro TrpThr Leu Ile 275 280 285 Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn GluThr Trp Glu 290 295 300 Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly GluPhe Trp Leu 305 310 315 Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln SerAsn Tyr Val 320 325 330 Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn LysHis Tyr Ile 335 340 345 Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr AsnTyr Thr Leu 350 355 360 His Leu Val Ala Ile Thr Gly Asn Val Pro Asn AlaIle Pro Glu 365 370 375 Asn Lys Asp Leu Val Phe Ser Thr Trp Asp His LysAla Lys Gly 380 385 390 His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly TrpTrp Trp His 395 400 405 Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys TyrAsn Lys Pro 410 415 420 Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly LeuSer Trp Lys 425 430 435 Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser ThrLys Met Leu 440 445 450 Ile His Pro Thr Asp Ser Glu Ser Phe Glu 455 460

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence having at least about 90% sequence identity with the amino acidsequence of native human NL-5 (SEQ ID NO: 4), and having the ability toinduce vascularization.
 2. A composition comprising a polypeptideaccording to claim 1, in association with a carrier.
 3. A conjugatecomprising a polypeptide according to claim 1, fused to a therapeutic orcytotoxic agent.
 4. The conjugate of claim 3 wherein the therapeuticagent is a toxin, a TIE ligand, or a member of the vascular endothelialgrowth factor (VEGF) family.
 5. The polypeptide of claim 1 comprisingthe fibrinogen-like region of native sequence human NL-5 of SEQ ID NO:4.
 6. A composition comprising a polypeptide according to claim
 5. 7. Aconjugate comprising a polypeptide according to claim
 5. 8. An isolatedpolypeptide comprising the amino acid sequence of native human NL-5 (SEQNO: 4), and having the ability to induce vascularization.
 9. Acomposition comprising an isolated polypeptide comprising the amino acidsequence of native human NL-5 (SEQ ID NO: 4), and having the ability toinduce vascularization.
 10. A conjugate comprising an isolatedpolypeptide comprising the amino acid sequence of native human NL-5 (SEQID NO: 4), and having the ability to induce vascularization.
 11. Anisolated polypeptide comprising the amino acid sequence of native humanNL-5 (SEQ ID NO: 4).
 12. A composition comprising a polypeptideaccording to claim
 11. 13. A conjugate comprising a polypeptideaccording to claim 11.